The authors meet criteria for authorship as recommended by the International Committee of Medical Journal Editors (ICMJE). The authors received no direct compensation related to the development of the manuscript. Writing assistance was provided by Ken Sutor, BSc, of GeoMed, an Ashfield company, part of UDG Healthcare plc, which was contracted and funded by Boehringer Ingelheim Pharmaceuticals, Inc. (BIPI). BIPI was given the opportunity to review the manuscript for medical and scientific accuracy as well as intellectual property considerations.
The authors received no direct compensation related to the development of the video. Medical writing support for the video script was provided by Leon Newman, PhD, of GeoMed, an Ashfield company, which was contracted and funded by Boehringer Ingelheim Pharmaceuticals, Inc. (BIPI). BIPI was given the opportunity to review the final video for medical and scientific accuracy as well as intellectual property considerations.

1. HRCT scanning
<ol>
	<li>Perform volumetric HRCT acquisition scanning of the chest36. Contrast agents are not required36,37:</li>
	<li>Obtain the following acquisitions with parameters shown in Table 136,37.
	<ol>
		<li>Acquire a supine inspiratory scan (volumetric) from the lung apices to the lung base.</li>
		<li>Acquire a supine expiratory scan (sequential with 10&#8211;20 mm gaps) from 2 cm below the lung apices to the lung base.</li>
		<li>Acquire a prone inspiratory optional (sequential with 10&#8211;20 mm gaps) from the carina to the lung base.</li>
	</ol>
	</li>
	<li>Give breathing instructions to the patient before each acquisition36,37. For an inspiratory scan, say &#8220;Take in a deep breath&#8230;.and let it out. Take in another deep breath&#8230;.and let it out. Take in another deep breath, and hold your breath in. Keep holding your breath&#8221;37.</li>
	<li>Obtain inspiratory scans at full inspiration35,36.</li>
	<li>Use the thinnest collimation, shortest rotation time and highest pitch to ensure that motion-free images are obtained36. Suggested scanning parameters are detailed in Table 137.</li>
	<li>For optimal quality of volumetric scans, obtain thin section (&#60;2 mm) images with high-spatial resolution reconstruction35,36.</li>
	<li>Review scans immediately after acquisition and repeat if either motion artifact is present or inadequate inspiration has occurred37.</li>
</ol>
2. Reporting
<ol>
	<li>Prepare an interpretive report.</li>
	<li>Share the report and HRCT images with the patient&#8217;s care team and add them to the patient&#8217;s medical records.</li>
</ol>

Jonathan Chung declares no potential conflicts of interest with respect to the described research, authorship, and/or publication of this article; Christopher Walker declares the receipt of royalties from Amirsys (Elsevier); and Stephen Hobbs declares the receipt of royalties from Elsevier and Wolters Kluwer Health. Open Access publication of this article is sponsored by Boehringer Ingelheim Pharmaceuticals, Inc.

While HRCT is currently the definitive imaging method for diagnosing and assessing SSc-ILD, it uses ionizing radiation and is relatively expensive. Chest radiographs may be undertaken instead, although these do not facilitate differential diagnosis to the same extent as HRCT, and a normal chest radiograph does not eliminate the possibility of ILD. Perhaps the best use of chest radiographs is to monitor for progressive disease between HRCT scans and for the exclusion of complicating disease, such as infectious pneumonia, in the setting of acute worsening of symptoms.
A perceived limitation of HRCT is radiation exposure. As described earlier, new methods of conducting CT scans may enable radiation exposure to be reduced31, and furthermore, current CT scanners provide an array of advanced techniques that offer the possibility in the future to lower radiation exposure to nearly chest radiograph levels. Alternatively, imaging methods such as MRI or lung ultrasound could potentially be used to avoid exposing the patient to radiation in the future32,72,73,74. We believe that, while there are risk-benefit considerations associated with imaging utilization, the advantages of CT in diagnosis and patient management far outweigh the potential risks.
Imaging data, particularly HRCT, provide arguably the most important information to enable diagnosis of SSc-ILD. Detailed consideration of the patterns and features of HRCT scans is usually sufficient to distinguish SSc-ILD from other lung diseases, with the benefit of avoiding the need for an invasive biopsy procedure.
Visual assessment of HRCT scans introduces a degree of subjectivity and the possibility of inter-observer variability. Computer-based methods of HRCT scan interpretation have been investigated as a possible approach to improving accuracy63,75. For example, quantitative approaches to the assessment of lung fibrosis or the extent of disease may be used to assess treatment response69,71,76. However, these methods are not widely used in daily clinical practice at this time.
We hope the information presented in this manuscript will serve as a practical guide to assist physicians in using HRCT scans for diagnosing SSc-ILD and determining prognosis. Improved methods for obtaining images and for interpreting scans have the potential to reduce patients&#8217; exposure to radiation and improve diagnostic/prognostic accuracy.

Systemic sclerosis (SSc) is a complex, heterogeneous, autoimmune disease. It may be manifested as vasculopathy, Raynaud&#8217;s phenomenon and fibrosis of the skin and internal organs1. SSc is classified into subtypes as follows: limited cutaneous, diffuse cutaneous, sine scleroderma (without skin involvement), and SSc overlap syndrome1.
SSc is not inherited in Mendelian fashion, but genetic factors appear to influence susceptibility to the disease. Incidence rates differ between ethnic groups and are increased among individuals with a family history of the disease2,3. Environmental risk factors also appear to exist, with high exposure to silica or organic solvents appearing to increase the occurrence of SSc4. The global prevalence of SSc is around 1 in 10,0001. More females than males are affected by SSc, with reported female:male ratios ranging between 3:1 and 8:1, and the age group with the highest incidence of the disease is 45&#8211;54 years5.
The lung is the second most commonly affected visceral organ in patients with SSc6. There are two main pulmonary manifestations of SSc: interstitial lung disease (ILD), and pulmonary hypertension7. ILD is usually fibrotic; it occurs in approximately 80% of patients with SSc and is more common in diffuse cutaneous scleroderma than in the limited form of the disease1,8. Pulmonary hypertension may manifest as isolated pulmonary arterial hypertension (PAH, which has a prevalence of 13&#8211;35% in SSc) or pulmonary hypertension resulting from left ventricular involvement/diastolic dysfunction or ILD/hypoxemia7. Antibody profiles differ between patients with SSc-ILD and those with SSc-PAH. For example, the presence of anti-Scl-70 antibodies is associated with SSc-ILD8, while anticentromere antibodies are more common in SSc patients with PAH than in those without PAH9.
The symptoms of SSc-ILD include dyspnea, coughing, chest pain, and exercise limitation. ILD is a major contributor to morbidity in SSc10,11,12. As a consequence, annual all-cause healthcare costs have been reported to be higher in patients with SSc-ILD than in those with SSc and no ILD: $31,285&#8211;55,446 versus $18,513&#8211;23,268, respectively13.
SSc-ILD is the leading cause of mortality in patients with SSc, accounting for 30&#8211;35% of deaths in this group10,14. Median survival among patients with SSc-ILD has been reported to be 5&#8211;8 years10,15; by comparison, approximately 76% of the overall population with SSc survive for more than 10 years from disease onset16. Significant predictors of mortality in SSc-ILD include age, forced vital capacity (FVC), baseline diffusing capacity of the lung for carbon monoxide (DLCO), extent of disease on high-resolution computed tomography (HRCT), presence of pulmonary hypertension and levels of Krebs von den Lungen 6 (KL-6) antigen17,18.
Early diagnosis is important to enable treatment to be administered with minimal delay and, in patients with a progressive phenotype, disease progression may potentially be slowed. However, diagnosing SSc-ILD is challenging because non-specific symptoms of cough, dyspnea, and fatigue can be mistaken for other aspects of SSc, such as cardiac disease and musculoskeletal involvement. Evaluations for diagnosing ILDs include: clinical presentation, history, smoking status, lung function, imaging, and in some cases, lung biopsy. Affirmation of SSc-ILD diagnosis requires several investigations, which are often used in combination19. The most frequently used assessments include pulmonary function tests and HRCT20,21,22,23. Other imaging methods, such as chest radiography and radiation-sparing imaging (e.g., magnetic resonance imaging [MRI], lung ultrasound) may also be employed22. Pulmonary function tests are used to assess the severity of the ILD and monitor its course. However, the use of pulmonary function tests alone is of limited use for diagnosing SSc-ILD24,25. HRCT of the chest is viewed as the most sensitive non-invasive means of facilitating differential diagnosis of SSc-ILD19. Baseline HRCT results, as well as changes over time, can be used to predict the future course of lung disease and potential response to therapy26.
Exposure to radiation with HRCT is sometimes considered as a limiting factor for regular screening27,28; limiting the number of slices is a potential method for reducing the radiation risk, and the dose may also be reduced by decreasing either the voltage or the current29,30,31. Alternatively, different assessment methods may be considered. For example, MRI appears to have some potential for evaluation and follow-up of ILD patients22. In one study using T2-weighted MRI images with respiratory synchronization, HRCT was performed in parallel as the &#8216;gold-standard&#8217; assessment; 100% sensitivity and 60% specificity were reported with MRI for determining the presence of ILD32. Similar agreement between MRI and HRCT in the detection and categorization of ILD was reported in another study33. Despite the promising results, MRI is currently a research methodology and it is not yet ready for generalized clinical use.
Here, we provide a practical overview of the interpretation of imaging results, with a focus on HRCT, for diagnosing lung involvement in SSc, determining prognosis, and also exploring future developments that may improve imaging methods and interpretation of results. HRCT images from representative cases are included in the paper.

<table border="0" cellpadding="0" cellspacing="0" style="border-collapse:
 collapse;width:500pt" width="666">
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt;width:162pt" width="216">CT scanners</td>
			<td class="xl65" style="width:92pt" width="122">Philips</td>
			<td class="xl65" style="width:121pt" width="161">NA</td>
			<td class="xl66" style="width:125pt" width="167">Multiple</td>
		</tr>
	</tbody>
</table>

imaging features of systemic sclerosis-associated interstitial lung disease

Early diagnosis of systemic sclerosis-related interstitial lung disease (SSc-ILD) is important to enable treatment to be administered with minimal delay. However, diagnosing SSc-ILD is challenging because key symptoms are non-specific. High-resolution computed tomography (HRCT) of the chest is recognized as a sensitive imaging method for diagnosing and assessing SSc-ILD. Exposure of patients to ionizing radiation may be considered as a limitation, although methodological steps may be taken to moderate this. We present practical recommendations for performing HRCT scans and interpreting the results. Key features of SSc-ILD on HRCT include a non-specific interstitial pneumonia (NSIP) pattern with peripheral ground-glass opacities and extensive traction bronchiectasis. Despite similarities between SSc-ILD and idiopathic pulmonary fibrosis (IPF), HRCT can be used to differentiate between these conditions: in SSc-ILD compared with IPF, there is a greater proportion of ground-glass opacity and fibrosis is less coarse. A dilated, air-filled esophagus with diameter &#62;10 mm, suggestive of esophageal dysmotility is commonly seen in SSc-ILD. Pulmonary artery size greater than the adjacent ascending aorta suggests coexistent pulmonary hypertension. Nodules must be monitored due to the increased risk of lung cancer. A large extent of disease on HRCT (&#8805;20%) or a high fibrosis score suggests an increased risk of mortality. HRCT is central to diagnosing SSc-ILD, and serial assessments can be helpful in monitoring disease progression or treatment response.

Diagnosis 
Key features of SSc-ILD on HRCT commonly include a non-specific interstitial pneumonia (NSIP) pattern with peripheral ground-glass opacities and extensive traction bronchiectasis (Figure 1 and Figure 2). Ground-glass opacities have a broad etiology and are often non-specific40,41,42. Central predominance or peripheral distribution with subpleural sparing is highly suggestive of NSIP (Figure 3).
Typically, ILD patterns in HRCT images include reticulations with architectural distortion resulting in traction bronchiectasis/bronchiolectasis (consistent with a fibrotic form of NSIP). Indeed traction bronchiectasis and traction bronchiolectasis are often the predominant features of SSc-ILD (Figure 4)43. Additional findings may include honeycombing (Figure 5; more common in limited forms of SSc), interlobular septal thickening and intralobular lines, and micronodules40,44. Honeycombing refers to clustered cystic airspaces of typically consistent diameter (~3&#8211;10 mm) with thick, well defined walls31. Honeycombing and traction bronchiectasis are key features of usual interstitial pneumonia (UIP) on HRCT. Although this pattern is most commonly associated with idiopathic pulmonary fibrosis (IPF), the prototype fibrosing ILD with a progressive phenotype, it can sometimes be seen in patients with SSc-ILD10. Recently, several signs have been identified in patients with connective tissue disease-related ILD (including SSc-ILD) and the UIP pattern on HRCT, but not in those with IPF. These are the straight edge sign (i.e., isolation of fibrosis to the lung bases with sharp demarcation in the craniocaudal plane without substantial extension along the lateral margins of the lungs on coronal images), the honeycombing predominant (or exuberant) sign (&#62;70% of fibrotic portions of the lung), and the anterior upper lobe sign (i.e., concentration of fibrosis within the anterior aspect of the upper lobes, with relative sparing of the other aspects of the upper lobes, and concomitant lower lobe involvement)45. The straight edge sign has also been associated with NSIP pathology46, which is the main CT pattern in SSc-ILD10.
Dilated air-filled esophagus is frequently observed in patients with SSc (Figure 6)47,48,49 and in patients with SSc-ILD47,48. While there is no accepted upper age limit where a dilated esophagus may no longer help to differentiate SSc-ILD and IPF, a dilated esophagus may be more difficult to interpret in patients over age 65 due to increasing incidence of esophageal motility disorders. Mediastinal&#160;lymphadenopathy (usually reactive), in which the short axis of the lymph node exceeds 10 mm, is also often observed in patients with SSc-ILD47,50. Pulmonary artery size greater than the adjacent ascending aorta suggests coexistent pulmonary hypertension (Figure 6), even in patients without fibrotic lung disease51,52,53. Areas of consolidation suggest superimposed infection, aspiration, organizing pneumonia, hemorrhage or malignancy. Nodules must be monitored due to the increased risk for lung cancer in SSc-ILD7; the most common primary cancer to arise in patients with SSc-ILD is adenocarcinoma7,54.
SSc-ILD shares a number of clinical, mechanistic, and pathological similarities with IPF15,55. However, some radiologic features allow the differentiation of these two ILDs15,45. In SSc-ILD, compared with IPF, there is a greater proportion of ground-glass opacity and fibrosis is less coarse. In cases of UIP in SSc, honeycombing may be observed in more than 70% of the fibrotic-lung tissue &#9472; the exuberant honeycombing sign56,57. In addition, the four-corners sign (also known as the anterior upper lobe sign) is significantly more common in SSc-ILD than in IPF; this is a pattern of inflammation and/or fibrosis focally or disproportionately involving the bilateral anterolateral upper lobes and posterosuperior lower lobes58.
Chest radiographs may initially detect ILD; however, they do not offer enough contrast resolution for reliable diagnosis. In chest radiographs from patients with SSc-ILD, the most frequent pattern is basal predominant reticulation59. Further features may include visible bronchiectasis, volume loss and honeycombing. As with HRCT, the presence of a dilated air-filled esophagus may be helpful in supporting the diagnosis of SSc-ILD47.
Prognosis 
Several different imaging findings have been shown to be associated with prognosis in SSc-ILD. Mortality risk has been reported to be higher in patients with a disease extent of at least 20% on HRCT (10-year survival was 43% versus 67%, respectively, in patients with disease extent above versus below the 20% threshold)60. Similarly, a high fibrosis score on HRCT (based on the extent of reticulation and honeycombing) has been associated with increased mortality61. Large esophageal diameters are associated with increased ILD severity and decreased DLCO48. Lung density and pulmonary artery diameter may potentially be used to predict the risk of pulmonary hypertension62. Computerized, quantitative CT parameters could also be harnessed to identify patients&#8217; risk of lung function decline or mortality. One study suggested that the extent of ILD, quantified from HRCT, could be used to predict the decline in FVC over 12 months63. In another study, quantitative chest CT parameters provided mortality risk results that were consistent with clinical prediction models64. Despite their apparent potential, imaging-based biomarkers are currently best considered at a population level as their clinical utility in individual patients has not been established.
Treatment response 
Cyclophosphamide and mycophenolate mofetil provide modest benefit in patients with SSc-ILD. In the landmark Scleroderma Lung Study I, cyclophosphamide treatment led to slower progression of fibrosis compared with placebo65. More recently, the Scleroderma Lung Study II reported similar efficacy and improved tolerability with mycophenolate mofetil in comparison with cyclophosphamide66. Based upon positive SENSCIS&#174;&#160;trial results67, nintedanib became the first US Food and Drug Administration-approved treatment to slow the rate of decline in lung function in patients with SSc-ILD. However, there remains a need for improved treatment options for patients with SSc-ILD. Therapies currently being investigated include monoclonal antibodies (e.g. rituximab, abituzumab), antifibrotic agents (e.g., pirfenidone), the direct thrombin inhibitor dabigatran, the proteasome inhibitor bortezomib, and hematopoietic stem cell transplantation19,68.
Serial HRCT scans showing disease progression in a patient with SSc-ILD 
HRCT assessments performed at different timepoints may be used to investigate disease progression. Figure 7 shows two sets of axial and coronal chest HRCT images taken 10 years apart in a patient with SSc-ILD. The initial axial and coronal images (Figure 7A and B) from chest HRCT show basilar predominant ground-glass opacity and reticulation with mild traction bronchiectasis and subpleural sparing consistent with NSIP in this patient with SSc. The latter set of images (Figure 7C and&#160;D) taken 10 years later, show increased reticulation and traction bronchiolectasis at the lung bases with decrease in ground-glass opacity on axial and coronal (Figure 7C and&#160;D) images from chest CT consistent with mild worsening of pulmonary fibrosis. Serial HRCT scans can also be used to monitor treatment response69,70,71; this was demonstrated in the Scleroderma Lung Study II, in which computer-aided diagnosis scores based on HRCT scans were used to compare the efficacy of cyclophosphamide with mycophenolate mofetil in patients with SSc-ILD69.
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Phase</td>
			<td>Detector 
			collimation</td>
			<td>Voltage 
			(kVp)</td>
			<td>Current (mAs)</td>
			<td>Scan 
			interval</td>
			<td>Pitch</td>
			<td>Rotation</td>
			<td>Tube current 
			modulation</td>
		</tr>
		<tr>
			<td>Supine inspiratory</td>
			<td>Helical 1.2 mm</td>
			<td>120 (may be lowered)</td>
			<td>230 (may be lowered)</td>
			<td>N/A</td>
			<td>~1.0</td>
			<td>0.5 seconds or faster</td>
			<td>On</td>
		</tr>
		<tr>
			<td>Supine expiratory</td>
			<td>Axial 2 x 1.0 mm</td>
			<td>120</td>
			<td>150</td>
			<td>20 mm</td>
			<td>N/A</td>
			<td>1.0 seconds</td>
			<td>On</td>
		</tr>
		<tr>
			<td>Prone inspiratory</td>
			<td>Axial 2 x 1.0 mm</td>
			<td>120</td>
			<td>150</td>
			<td>20 mm</td>
			<td>N/A</td>
			<td>1.0 seconds</td>
			<td>On</td>
		</tr>
	</tbody>
</table>
Table 1: Computed tomography acquisition parameters37. kVp = kilovoltage peak;&#160;N/A = not applicable.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60300/60300fig1v2.jpg" /> 
Figure 1: Systemic sclerosis with a cellular NSIP pattern of disease. Axial (A), prone (B) and coronal (C) high-resolution computed tomography images all show extensive peripheral and basal predominant ground-glass opacities; these are typical observations with NSIP. The lack of traction bronchiectasis is suggestive of a cellular NSIP pattern of disease. NSIP = non-specific interstitial pneumonia. <a href="https://www.jove.com/files/ftp_upload/60300/60300fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60300/60300fig2v2.jpg" /> 
Figure 2: Systemic sclerosis with a fibrotic non-specific interstitial pneumonia pattern of disease. Axial computed tomography image shows extensive, basal-predominant ground-glass opacities with associated traction bronchiectasis. Notably, the esophagus shows marked dilation; this is typical of scleroderma. <a href="https://www.jove.com/files/ftp_upload/60300/60300fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60300/60300fig3v3.jpg" /> 
Figure 3: Systemic sclerosis with a fibrotic NSIP pattern. Axial high-resolution computed tomography images (A and B) show extensive ground-glass opacities, reticulation, architectural distortion and traction bronchiectasis. Notably, subpleural sparing is apparent; this is typical of NSIP and is seen in about 50% of all cases. NSIP = non-specific interstitial pneumonia. <a href="https://www.jove.com/files/ftp_upload/60300/60300fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60300/60300fig4v3.jpg" /> 
Figure 4: Systemic sclerosis with exuberant traction bronchiectasis. Axial (A) and coronal (B) high-resolution computed tomography images show extensive middle and lower lung zone predominant traction bronchiectasis. While this may be mistaken for honeycombing, the cystic areas connect with each other and spare the immediate subpleural lung; this is typical of bronchiectasis. <a href="https://www.jove.com/files/ftp_upload/60300/60300fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60300/60300fig5v3.jpg" /> 
Figure 5: Systemic sclerosis with a UIP pattern of lung fibrosis. Axial (A) and coronal (B) computed tomography images show peripheral and basal predominant honeycombing and traction bronchiectasis in keeping with the typical UIP pattern of lung fibrosis. Note the dilated esophagus (attributable to scleroderma) and the &#8216;exuberant&#8217; honeycombing (suggestive of ILD related to connective tissue disease rather than idiopathic pulmonary fibrosis). UIP = usual interstitial pneumonia. <a href="https://www.jove.com/files/ftp_upload/60300/60300fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60300/60300fig6v3.jpg" /> 
Figure 6: Systemic sclerosis with pulmonary hypertension and dilated esophagus. Contrast-enhanced chest computed tomography shows marked enlargement of the pulmonary trunk, with a larger measurement than the adjacent ascending aorta that suggests underlying pulmonary hypertension. The esophagus is markedly dilated; this is attributable to scleroderma. <a href="https://www.jove.com/files/ftp_upload/60300/60300fig6v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/60300/60300fig7v2.jpg" /> 
Figure 7: Serial chest HRCT images showing progression of pulmonary fibrosis in patient with SSc-ILD. Axial (A) and coronal (B) images from chest HRCT show basilar predominant ground-glass opacity and reticulation with mild traction bronchiectasis and subpleural sparing consistent with non-specific interstitial pneumonia in this patient with SSc.&#160;After 10 years, increased reticulation and traction bronchiolectasis at the lung bases with decrease in ground-glass opacity are observed on axial (C) and coronal (D) chest HRCT images, consistent with mild worsening of pulmonary fibrosis. HRCT = high-resolution computed tomography; SSc-ILD = systemic scleroderma-associated interstitial lung disease. <a href="https://www.jove.com/files/ftp_upload/60300/60300fig7v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Imaging Features of Systemic Sclerosis-Associated Interstitial Lung Disease at JoVE.com

Imaging Features of Systemic Sclerosis-Associated Interstitial Lung Disease

Here, we present practical recommendations for performing thoracic high-resolution computed tomography for diagnosing and assessing systemic sclerosis-related interstitial lung disease.

Early diagnosis of systemic sclerosis-related interstitial lung disease (SSc-ILD) is important to enable treatment to be administered with minimal delay. ...

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19.4K Views.  University of Chicago Medicine. Here, we present practical recommendations for performing thoracic high-resolution computed tomography for diagnosing and assessing systemic sclerosis-related interstitial lung disease.

Video: Imaging Features of Systemic Sclerosis-Associated Interstitial Lung Disease

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 (Fig.1). A 12 &mu;m thin tissue section is covered with a MALDI matrix, which facilitates desorption and ionization of intact peptides and proteins that can be detected with a mass analyzer, typically using a MALDI TOF/TOF mass spectrometer. Generally hundreds of peaks can be assessed in a single rat brain tissue section. In contrast to commonly used imaging techniques, this approach does not require prior knowledge of the molecules of interest and allows for unsupervised and comprehensive analysis of multiple molecular species while maintaining high molecular specificity and sensitivity2. Here we describe a MALDI IMS based approach for elucidating region-specific distribution profiles of neuropeptides in the rat brain of an animal model Parkinson's disease (PD). 
PD is a common neurodegenerative disease with a prevalence of 1% for people over 65 of age3,4. The most common symptomatic treatment is based on dopamine replacement using L-DOPA5. However this is accompanied by severe side effects including involuntary abnormal movements, termed L-DOPA-induced dyskinesias (LID)1,3,6. One of the most prominent molecular change in LID is an upregulation of the opioid precursor prodynorphin mRNA7. The dynorphin peptides modulate neurotransmission in brain areas that are essentially involved in movement control7,8. However, to date the exact opioid peptides that originate from processing of the neuropeptide precursor have not been characterized. Therefore, we utilized MALDI IMS in an animal model of experimental Parkinson's disease and L-DOPA induced dyskinesia. 
MALDI imaging mass spectrometry proved to be particularly advantageous with respect to neuropeptide characterization, since commonly used antibody based approaches targets known peptide sequences and previously observed post-translational modifications. By contrast MALDI IMS can unravel novel peptide processing products and thus reveal new molecular mechanisms of neuropeptide modulation of neuronal transmission. While the absolute amount of neuropeptides cannot be determined by MALDI IMS, the relative abundance of peptide ions can be delineated from the mass spectra, giving insights about changing levels in health and disease. In the examples presented here, the peak intensities of dynorphin B, alpha-neoendorphin and substance P were found to be significantly increased in the dorsolateral, but not the dorsomedial, striatum of animals with severe dyskinesia involving facial, trunk and orolingual muscles (Fig. 5). Furthermore, MALDI IMS revealed a correlation between dyskinesia severity and levels of des-tyrosine alpha-neoendorphin, representing a previously unknown mechanism of functional inactivation of dynorphins in the striatum as the removal of N-terminal tyrosine reduces the dynorphin's opioid-receptor binding capacity9. This is the first study on neuropeptide characterization in LID using MALDI IMS and the results highlight the potential of the technique for application in all fields of biomedical research.

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson&#039;s Disease

Dopamine replacement pharmacotherapy using L-DOPA is the most commonly used symptomatic treatment of Parkinson&#x2019;s disease, but is accompanied by side effects including involuntary abnormal movements, termed dyskinesia 1. Here, a protocol for MALDI imaging mass spectrometry is presented that detects changes in rat brain neuropeptide levels related to dyskinesia.

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Diagnostic Ultrasound Imaging of Mouse Diaphragm Function

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive surgical procedures. Although this is an effective method of assessing in vitro diaphragm activity, it involves non-survival surgery. The application of non-invasive ultrasound imaging as an in vivo procedure is beneficial since it not only reduces the number of animals sacrificed, but is also suitable for monitoring disease progression in live mice. Thus, our ultrasound imaging method may likely assist in the development of novel therapies that alleviate muscle injury induced by various respiratory diseases. Particularly, in clinical diagnoses of obstructive lung diseases, ultrasound imaging has the potential to be used in conjunction with other standard tests to detect the early onset of diaphragm muscle fatigue. In the current protocol, we describe how to accurately evaluate diaphragm contractility in a mouse model using a diagnostic ultrasound imaging technique.

Diagnostic ultrasound imaging has proven to be effective in diagnosing various respiratory diseases in human and animal subjects. We demonstrate a comprehensive ultrasound protocol utilized by Dr. Zuo's lab to analyze diaphragm kinetics specifically in mouse models. This is also a non-invasive research technique which can provide quantitative information on mouse respiratory muscle function.

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive ...

Tracking the Mammary Architectural Features and Detecting Breast Cancer with Magnetic Resonance Diffusion Tensor Imaging

Breast cancer is the most common cause of cancer among women worldwide. Early detection of breast cancer has a critical role in improving the quality of life and survival of breast cancer patients. In this paper a new approach for the detection of breast cancer is described, based on tracking the mammary architectural elements using diffusion tensor imaging (DTI). 
The paper focuses on the scanning protocols and image processing algorithms and software that were designed to fit the diffusion properties of the mammary fibroglandular tissue and its changes during malignant transformation. The final output yields pixel by pixel vector maps that track the architecture of the entire mammary ductal glandular trees and parametric maps of the diffusion tensor coefficients and anisotropy indices. 
The efficiency of the method to detect breast cancer was tested by scanning women volunteers including 68 patients with breast cancer confirmed by histopathology findings. Regions with cancer cells exhibited a marked reduction in the diffusion coefficients and in the maximal anisotropy index as compared to the normal breast tissue, providing an intrinsic contrast for delineating the boundaries of malignant growth. Overall, the sensitivity of the DTI parameters to detect breast cancer was found to be high, particularly in dense breasts, and comparable to the current standard breast MRI method that requires injection of a contrast agent. Thus, this method offers a completely non-invasive, safe and sensitive tool for breast cancer detection.

We describe how to obtain parametric and vector maps of the diffusion tensor of the breast using magnetic resonance imaging. The protocol and final output following imaging processing are tailored for tracking breast architectural features and detecting breast malignancy.

Breast cancer is the most common cause of cancer among women worldwide. Early detection of breast cancer has a critical role in improving the quality of ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting and sustaining this phenotype is critically important in normal alveolar growth and gas exchange throughout life. Several in vitro studies have characterized the role of key regulatory proteins, signaling molecules, and steroid hormones that can influence the rate of lung fluid clearance. However, in vivo examinations must be performed to evaluate whether these regulatory factors play important physiological roles in regulating perinatal lung liquid absorption. As such, the utilization of real time X-ray imaging to determine perinatal lung fluid clearance, or pulmonary edema, represents a technological advancement in the field. Herein, we explain and illustrate an approach to assess the rate of alveolar lung fluid clearance and alveolar flooding in C57BL/6 mice at post natal day 10 using X-ray imaging and analysis. Successful implementation of this protocol requires prior approval from institutional animal care and use committees (IACUC), an in vivo small animal X-ray imaging system, and compatible molecular imaging software.

We present a protocol to assess the rate of alveolar fluid clearance or pulmonary edema in neonatal mouse lung using X-ray imaging technology.

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting ...

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular ...

Generation of Human 3D Lung Tissue Cultures (3D-LTCs) for Disease Modeling

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) used as 3D lung tissue cultures (3D-LTCs) represent an elegant and biologically highly relevant 3D cell culture model, which highly resemble in situ tissue due to their complexity, biomechanics and molecular composition. Tissue slicing is widely applied in various animal models. 3D-LTCs derived from human PCLS can be used to analyze responses to novel drugs, which might further help to better understand the mechanisms and functional effects of drugs in human tissue. The preparation of PCLS from surgically resected lung tissue samples of patients, who experienced lung lobectomy, increases the accessibility of diseased and peritumoral tissue. Here, we describe a detailed protocol for the generation of human PCLS from surgically resected soft-elastic patient lung tissue. Agarose was introduced into the bronchoalveolar space of the resectates, thus preserving lung structure and increasing the tissue&#39;s stiffness, which is crucial for subsequent slicing. 500 &#181;m thick slices were prepared from the tissue block with a vibratome. Biopsy punches taken from PCLS ensure comparable tissue sample sizes and further increase the amount of tissue samples. The generated lung tissue cultures can be applied in a variety of studies in human lung biology, including the pathophysiology and mechanisms of different diseases, such as fibrotic processes at its best at (sub-)cellular levels. The highest benefit of the 3D-LTC ex vivo model is its close representation of the in situ human lung in respect of 3D tissue architecture, cell type diversity and lung anatomy as well as the potential for assessment of tissue from individual patients, which is relevant to further develop novel strategies for precision medicine.

Generation of Human 3D Lung Tissue Cultures 3D-LTCs for Disease Modeling

Here, we present a protocol for the preparation of agarose-filled human precision-cut lung slices from resected patient tissue that are suitable for generating 3D lung tissue cultures to model human lung diseases in biological and biomedical studies.

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) ...

Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of the fresh gas entering a lung region divided by the region&#8217;s end-expiratory volume &#8213; in the human lung, using only inhaled oxygen as a contrast agent. Regional quantification of specific ventilation has the potential to help identify areas of pathologic lung function. Oxygen in solution in tissue shortens the tissue&#8217;s longitudinal relaxation time (T1), and thus a change in tissue oxygenation can be detected as a change in T1-weighted signal with an inversion recovery acquired image. Following an abrupt change between two concentrations of inspired oxygen, the rate at which lung tissue within a voxel equilibrates to a new steady-state reflects the rate at which resident gas is being replaced by inhaled gas. This rate is determined by specific ventilation. To elicit this sudden change in oxygenation, subjects alternately breathe 20-breath blocks of air (21% oxygen) and 100% oxygen while in the MRI scanner. A stepwise change in inspired oxygen fraction is achieved through use of a custom three-dimensional (3D)-printed flow bypass system with a manual switch during a short end-expiratory breath hold. To detect the corresponding change in T1, a global inversion pulse followed by a single shot fast spin echo sequence was used to acquire two-dimensional T1-weighted images in a 1.5&#160;T MRI scanner, using an eight-element torso coil. Both single slice and multi-slice imaging are possible, with slightly different imaging parameters. Quantification of specific ventilation is achieved by correlating the time-course of signal intensity for each lung voxel with a library of simulated responses to the air/oxygen stimulus. SVI estimations of specific ventilation heterogeneity have been validated against multiple breath washout and proved to accurately determine the heterogeneity of the specific ventilation distribution.

Specific ventilation imaging is a functional magnetic resonance imaging technique that allows for quantification of regional specific ventilation in the human lung, using inhaled oxygen as a contrast agent. Here, we present a protocol to collect and analyze specific ventilation imaging data.

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of ...