Use of High Frequency Ultrasound to Monitor Cervical Lymph Node Alterations in Mice

<div><p>Cervical lymph node evaluation by clinical ultrasound is a non-invasive procedure used in diagnosing nodal status, and when combined with fine-needle aspiration cytology (FNAC), provides an effective method to assess nodal pathologies. Development of high-frequency ultrasound (HF US) allows real-time monitoring of lymph node alterations in animal models. While HF US is frequently used in animal models of tumor biology, use of HF US for studying cervical lymph nodes alterations associated with murine models of head and neck cancer, or any other model of lymphadenopathy, is lacking. Here we utilize HF US to monitor cervical lymph nodes changes in mice following exposure to the oral cancer-inducing carcinogen 4-nitroquinoline-1-oxide (4-NQO) and in mice with systemic autoimmunity. 4-NQO induces tumors within the mouse oral cavity as early as 19 wks that recapitulate HNSCC. Monitoring of cervical (mandibular) lymph nodes by gray scale and power Doppler sonography revealed changes in lymph node size eight weeks after 4-NQO treatment, prior to tumor formation. 4-NQO causes changes in cervical node blood flow resulting from oral tumor progression. Histological evaluation indicated that the early 4-NQO induced changes in lymph node volume were due to specific hyperproliferation of T-cell enriched zones in the paracortex. We also show that HF US can be used to perform image-guided fine needle aspirate (FNA) biopsies on mice with enlarged mandibular lymph nodes due to genetic mutation of Fas ligand (Fasl). Collectively these studies indicate that HF US is an effective technique for the non-invasive study of cervical lymph node alterations in live mouse models of oral cancer and other mouse models containing cervical lymphadenopathy.</p></div

Image-guided fine needle biopsy of Fasl mandibular lymph nodes.

<p><b>A.</b> Transverse section of a Fasl mouse neck imaged with HF US. The enlarged cervical mandibular node is evident as an oval hypoechoic region near the skin surface (circumscribed in yellow). Scale bar  = 1 mm. <b>B.</b> Frames from fine needle biopsy of a Fasl mandibular node guided by HF US. Images show the position of the sampling hyperechoic needle tip prior to cervical skin penetration (<i>left</i>), position of the needle during tissue removal (<i>middle</i>), and following needle withdrawal (<i>right</i>). Note the break in the skin following needle withdrawal (arrow). The angle and trajectory of the dorsal needle surface is denoted by the yellow dotted line. Scale bar  = 1 mm. The entire procedure is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100185#pone.0100185.s003" target="_blank">Video S1</a>. <b>C.</b> Examples of lymph tissue obtained by HF US guided FNA of a Fasl cervical mandibular node following staining and processing by cytospin. Scale bar  = 100 µm. LT; lymph tissue, RF; reticular fibers, L; individual lymphocytes.</p

4-NQO exposure induces precancerous alterations in mouse mandibular lymph nodes.

<p><b>A–C.</b> Images of dissected H&E and whole animal HF US (ultrasound) mandibular lymph nodes from representative age-matched (AM) control (<b>A</b>), 4-NQO-treated (28 wk) (<b>B</b>) and Fasl (<b>C</b>) mice. Lymph node borders in the HF US images are indicated in yellow. Vascular flow identified by power Doppler imaging is shown in red. Power Doppler flow dynamics for each condition are visualized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100185#pone.0100185.s004" target="_blank">Video S2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100185#pone.0100185.s006" target="_blank">S4</a>. H&E scale bar  = 250 µm, ultrasound scale bar  = 1 mm. CP, Cheek Pouch. <b>D&E.</b> Analysis of lymph nodes by HF US. 4-NQO treated mice at 0 and 28 wk were imaged after 8 week 4-NQO treatment and study end point. B6 age-matched (AM) Ctl 0 and 28 wk mice were imaged at the same age as 4-NQO treated mice. The Fasl lymph node data is included for comparison. <b>D.</b> 4-NQO exposure induces increased mandibular lymph node volume. <b>E.</b> 4-NQO exposure increases vascular flow in mandibular nodes. N = 6 lymph nodes from 3 mice per group, except for the controls, where N = 8 lymph nodes were analyzed from 4 mice. Box and whisker plots show minimum, 25^th, median, 75^th and maximum values, respectively. *, p≤0.05.</p

Mapping of mouse cervical lymph nodes by high frequency ultrasound.

<p><b>A.</b> Overview image of the HF US platform for cervical lymph node evaluation. An anesthetized B6 mouse is shown positioned on the Vevo 2100 heated imaging platform with the ventral side exposed. Each paw is tapped to a monitoring electrode and the rectal probe (blue) secured to the stage. The transducer (white) is positioned over the ventral neck area. <b>B.</b> Diagram showing relative locations of murine cervical lymph nodes. Individual neck sections visualized by HF US imaging and histology are indicated by dashed lines. Arrows denote specific positions of each mapped section relative to corresponding ultrasound and histology images. Each imaged anatomical location is numbered. M, mandibular node. AM, accessory mandibular node. SP, superficial parotid node. <b>C.</b> Serial transverse sections of the mouse neck imaged by HF US corresponding to the indicated anatomic regions in (B). <b>D.</b> Transverse cervical H&E stained histological sections corresponding to the HF US sections in (C). Arrows labeled “2” denote mandibular node as diagrammed in B. Scale bar  = 1 mm. CP, cheek pouch. VT, ventral tongue. DT, dorsal tongue. E, esophagus.</p

4-NQO treatment induces paracortical/T-cell zone hyperplasia in mandibular lymph nodes.

<p>Representative examples of H&E stained, dissected mandibular lymph nodes from age-matched (<b>A</b>) control and (<b>B</b>) 4-NQO-treated (28 wk) mice. T-cell zone expansions in each node are circumscribed in yellow. Scale bar  = 250 µm. <b>C.</b> Distribution of nodal paracortical/T-cell zone hyperplasia. Mandibular lymph nodes were pathologically scored and grouped according to relative scale of T-cell zone involvement, using the following scale: None, absent to focal limited expansion; Modest, multifocal or focal up to moderate expansion; Robust, multifocal moderate expansion and/or confluence of paracortical subregions.</p