Clearing-induced tisssue shrinkage: A novel observation of a thickness size effect

The use of clearing agents has provided new insights in various fields of medical research (developmental biology, neurology) by enabling examination of tissue architecture in 3D. One of the challenges is that clearing agents induce tissue shrinkage and the shrinkage rates reported in the literature are incoherent. Here, we report that for a classical clearing agent, benzyl-alcohol benzyl-benzoate (BABB), the shrinkage decreases significantly with increasing sample size, and present an analytical formula describing this.

Splenic Micronodular T-Cell/Histiocyte-Rich Large B-Cell Lymphoma: The Corticosteroid Pretreatment Hypothesis

Splenic micronodular T-cell/histiocyte-rich large B-cell lymphoma is derived from diffuse large B-cell lymphoma N.O.S., perhaps with some affinity with nodal T-cell/histiocyte-rich large B-cell lymphoma. Of note, in contrast with the latter, the only lymph nodes involved in association with the splenic micronodular pattern of the disease are the splenic hilar lymph nodes. The possibility that corticosteroids, when prescribed prior to splenectomy, cause histopathological and functional modulations, apoptosis, necrosis, tissue shrinkage, which may obscure the diagnostic morphological features of this variant lymphoma and cause and underdiagnosis of this condition. The indications for glucocorticoid therapy are either related to the lymphoma itself, or else to other comorbidities, like asthma and autoimmune disorders. We propose that patients with the splenic subset of the disease are likely to have been prescribed corticosteroids prior to histopathologic examination of the involved spleen, causing disparate morphologies. However, a reviewer might accidentally dismiss the corticosteroid pretreatment which is thus overlooked. Apoptosis, induced by corticosteroids, is hypothesized as the major mechanism initiating the histopathological and functional changes in the splenic micronodular variant of the lymphoma.

Reducing soft-tissue shrinkage artefacts caused by staining with Lugol’s solution

Diffusible iodine-based contrast-enhanced computed tomography (diceCT) is progressively used in clinical and morphological research to study developmental anatomy. Lugol’s solution (Lugol) has gained interest as an effective contrast agent; however, usage is limited due to extensive soft-tissue shrinkage. The mechanism of Lugol-induced shrinkage and how to prevent it is largely unknown, hampering applications of Lugol in clinical or forensic cases where tissue shrinkage can lead to erroneous diagnostic conclusions. Shrinkage was suggested to be due to an osmotic imbalance between tissue and solution. Pilot experiments pointed to acidification of Lugol, but the relation of acidification and tissue shrinkage was not evaluated. In this study, we analyzed the relation between tissue shrinkage, osmolarity and acidification of the solution during staining. Changes in tissue volume were measured on 2D-segmented magnetic resonance and diceCT images using AMIRA software. Partial correlation and stepwise regression analysis showed that acidification of Lugol is the main cause of tissue shrinkage. To prevent acidification, we developed a buffered Lugol’s solution (B-Lugol) and showed that stabilizing its pH almost completely prevented shrinkage without affecting staining. Changing from Lugol to B-Lugol is a major improvement for clinical and morphological research and only requires a minor adaptation of the staining protocol.

Splenic Micronodular T-Cell/Histiocyte-Rich Large B-Cell Lymphoma: The Corticosteroid Pretreatment Hypothesis

Splenic micronodular T-cell/histiocyte-rich large B-cell lymphoma is possibly derived from nodal T-cell/histiocyte-rich large B-cell lymphoma; however, a transition between the nodal and splenic micronodular forms has not been described to date. Of note, the only lymph nodes to be involved in association with the splenic micronodular pattern of the disease are the splenic hilar lymph nodes, and that, with partial involvement only. Kan et al, in their series of articles, have raised the possibility that corticosteroids, when prescribed prior to splenectomy, cause histopathological and functional modulations (apoptosis, necrosis, tissue shrinkage), which modify or even obscure the diagnostic morphological features. The indications for glucocorticoid therapy are either related to the suspected lymphoma, or else to other comorbidities, like asthma and autoimmune disorders. We propose that patients with the splenic, rather than nodal subset of the disease are likely to have been prescribed corticosteroids prior to histopathologic examination of the involved tissue, causing disparate morphologies in the spleen. Apoptosis, as induced by corticosteroids, is hypothesized as the major mechanism initiating the histopa-thological and functional changes in the splenic micronodular variant of our patients.

Radiofrequency Thermoablation On Ex Vivo Animal Tissues: Changes on Isolated Swine Thyroids

The use of Radiofrequency thermoablation (RFA) for treating large thyroid nodules is limited by the modest efficiency of the available systems in terms of volume of the ablation zones (AZs). This increases the risk of incomplete ablation of the nodule. Systems employing perfused electrodes have been developed to increase the volume of the AZ. Aim of this study is to compare the size of the AZ induced by RFA systems using internally cooled perfused vs. non-perfused electrodes in swine thyroids. RFAs were performed on 40 freshly isolated swine thyroids using both systems. The perfused system was tested using 0.9% saline, 7% and 18% hypertonic saline solutions. Energy delivery to the tissue was stopped when tissue conductivity dropped (real life simulations) and after an established time of 20 seconds (controlled duration). Following RFA, thyroids were transversally and longitudinally cut. Photographs were taken for macroscopic morphometry of the ablated zones before and after formalin fixation, to evaluate tissue shrinkage. Microscopic morphometry was performed on PAS stained sections. In real life simulation experiments, gross morphometry revealed that AZs produced with electrodes perfused using 7.0% saline are larger compared to isotonic saline. Microscopically, all the conditions tested using the perfused system produced larger AZs compared to non-perfused system after 20 seconds of RFA. In conclusion, the perfusion with 7.0% NaCl solution increased the electrical conductivity of the tissue in real life simulations, resulting in larger ablated areas compared to the use of isotonic saline.

Development of an Optimized Clearing Protocol to Examine Adipocyte Subpopulations in White Adipose Tissue

Organic solvent dibenzyl ether (DBE)-based protocols have been widely used in adipose tissue clearing. However, benzyl alcohol/benzyl benzoate (BABB)-based clearing has been shown to offer better transparency in other tissues. The addition of diphenyl ether (DPE) to BABB (BABB-D4) is often included to preserve fluorescent signals, but its effects on adipose tissue transparency and shrinkage have not been explored. Distinct adipocyte subpopulations contribute to its cellular composition and biological activity. Here, we compared clearing solvents to create an optimized clearing methodology for the study of adipocyte subpopulations. Adipose tissues were cleared with BABB, BABB-D4, and DBE, and post-clearing transparency and tissue shrinkage were measured. An optimized protocol, including BABB-D4 clearing, delipidation, and extensive immunofluorescence blocking steps, was created to examine the spatial distribution of Wt-1 positive progenitor-derived (Type-1) adipocytes in intact mesenteric fat. Both BABB and BABB-D4 lead to significantly increased tissue transparency with reduced tissue shrinkage compared to DBE-cleared adipose tissue. Type-1 adipocytes are found in a clustered distribution with predominant residence in fat associated with the ileum and colon. This paper details an optimized clearing methodology for adipose tissue with increased tissue transparency and reduced shrinkage, and therefore will be a useful tool for investigating adipose tissue biology.

An update to the Monro–Kellie doctrine to reflect tissue compliance after severe ischemic and hemorrhagic stroke

High intracranial pressure (ICP) can impede cerebral blood flow resulting in secondary injury or death following severe stroke. Compensatory mechanisms include reduced cerebral blood and cerebrospinal fluid volumes, but these often fail to prevent raised ICP. Serendipitous observations in intracerebral hemorrhage (ICH) suggest that neurons far removed from a hematoma may shrink as an ICP compliance mechanism. Here, we sought to critically test this observation. We tracked the timing of distal tissue shrinkage (e.g. CA1) after collagenase-induced striatal ICH in rat; cell volume and density alterations (42% volume reduction, 34% density increase; p < 0.0001) were highest day one post-stroke, and rebounded over a week across brain regions. Similar effects were seen in the filament model of middle cerebral artery occlusion (22% volume reduction, 22% density increase; p ≤ 0.007), but not with the Vannucci-Rice model of hypoxic-ischemic encephalopathy (2.5% volume increase, 14% density increase; p ≥ 0.05). Concerningly, this ‘tissue compliance’ appears to cause sub-lethal damage, as revealed by electron microscopy after ICH. Our data challenge the long-held assumption that ‘healthy’ brain tissue outside the injured area maintains its volume. Given the magnitude of these effects, we posit that ‘tissue compliance’ is an important mechanism invoked after severe strokes.

Comparison of Transparency and Shrinkage During Clearing of Insect Brains Using Media With Tunable Refractive Index

Improvement of imaging quality has the potential to visualize previously unseen building blocks of the brain and is therefore one of the great challenges in neuroscience. Rapid development of new tissue clearing techniques in recent years have attempted to solve imaging compromises in thick brain samples, particularly for high resolution optical microscopy, where the clearing medium needs to match the high refractive index of the objective immersion medium. These problems are exacerbated in insect tissue, where numerous (initially air-filled) tracheal tubes branching throughout the brain increase the scattering of light. To date, surprisingly few studies have systematically quantified the benefits of such clearing methods using objective transparency and tissue shrinkage measurements. In this study we compare a traditional and widely used insect clearing medium, methyl salicylate combined with permanent mounting in Permount (“MS/P”) with several more recently applied clearing media that offer tunable refractive index (n): 2,2′-thiodiethanol (TDE), “SeeDB2” (in variants SeeDB2S and SeeDB2G matched to oil and glycerol immersion, n = 1.52 and 1.47, respectively) and Rapiclear (also with n = 1.52 and 1.47). We measured transparency and tissue shrinkage by comparing freshly dissected brains with cleared brains from dipteran flies, with or without addition of vacuum or ethanol pre-treatments (dehydration and rehydration) to evacuate air from the tracheal system. The results show that ethanol pre-treatment is very effective for improving transparency, regardless of the subsequent clearing medium, while vacuum treatment offers little measurable benefit. Ethanol pre-treated SeeDB2G and Rapiclear brains show much less shrinkage than using the traditional MS/P method. Furthermore, at lower refractive index, closer to that of glycerol immersion, these recently developed media offer outstanding transparency compared to TDE and MS/P. Rapiclear protocols were less laborious compared to SeeDB2, but both offer sufficient transparency and refractive index tunability to permit super-resolution imaging of local volumes in whole mount brains from large insects, and even light-sheet microscopy. Although long-term permanency of Rapiclear stored samples remains to be established, our samples still showed good preservation of fluorescence after storage for more than a year at room temperature.

Degenerating Drosophila Larval Epidermal Cells Drive Thorax Closure

Adult thorax formation in Drosophila begins during pre-pupal development by fusion of its two contralateral progenitor halves, the heminotal epithelia (HE). HEs migrate and replace an underlying cell layer of thoracic larval epidermal cells (LECs) during a morphogenetic process called thorax closure. The LEC layer has so far been proposed to be a passive substrate over which HEs migrate before their zipping. By contrast, here we show that the pull forces generated within the LEC layer drive HE migration. During thorax closure, the LECs display actomyosin-mediated contraction, via enrichment of non-muscle myosin-II and actin, besides squamous-to-pseudostratified columnar epithelial transition and tissue shrinkage. This shrinkage of the LEC layer is further accompanied by cell extrusion and death, that prevent overcrowding of LECs, thereby promoting further shrinkage. The pull forces thus generated by the shrinking LEC layer are then relayed to the HEs by their mutual adhesions via βPS1 (Mys) and αPS3 (Scb) integrins. Suppression of cell death in the LEC layer by a gain of p35 leads to cell overcrowding, which impedes HE migration and zipping. Further, knockdown of sqh, the light chain of non-muscle myosin II, in LECs or integrins (mys or scb) in either the LEC layer or in the HEs, or both abrogate thorax closure. Mathematical modeling also reveals the biophysical underpinnings of the forces that drive this tissue closure process wherein a degenerating LEC layer mediates its succession by the future adult primodia. These essential principles of thorax closure appear ancient in origin and recur in multiple morphogenetic contexts and tissue repair.