Cryostat Sections Research Articles

Routine application of ex vivo confocal laser scanning microscopy with digital staining for examination of surgical margins in basal cell carcinomas.

Ex vivo confocal laser scanning microscopy (CLSM) allows histologic examination of native tissue based on tissue reflection and nuclear fluorescence staining. The newly introduced digital staining process almost perfectly mimics conventional hematoxylin and eosin (HE) slides. The aim was to evaluate the new method in clinical routine, with regard to quality of findings and time requirements, in the examination of surgical margins of basal cell carcinomas. 78 patients with 101 basal cell carcinomas were prospectively enrolled. Surgery was performed either with complete margin control (n = 60) or as elliptical excision (n = 41). Immediately after excision specimens were scanned with CLSM and then routinely processed by conventional histopathology. Blinded evaluation of images and slides was performed by a dermatopathologist. Basal cell carcinomas were excellently recognizable by CLSM directly after excision, and the use of digital staining did not require any adjustment of the examiner's visualization preferences. CLSM images showed a sensitivity of 73.6% and a specificity of 96.5% compared to conventional HE stained slides. Erroneous findings were often due to limited assessment potential in cases where the epidermis could not be fully visualized. CLSM with digital HE staining is very well suited to diagnose basal cell carcinomas and their incision margins even under routine conditions and thus represents a tissue-saving alternative to rapid cryostat sectioning.

Morphological and functional charakteristics of histogenetically related structures - producers of monoamines - in the local regulatory apparatus of the large duodenal papilla

Purpose of the study. Study of the morphology of open-type fluorescent cells and adrenergic nerve structures of the major duodenal papilla. Material and research methods. The material for the study was the area of the large duodenal papilla of 12 rabbits and 5 dogs. Fluorescent endocrine cells and adrenergic nerve structures were revealed by processing cryostat sections of the material with a gly-oxylic acid solution. Research results. It has been established that the fluorescent cells of the diffuse endocrine (APUD) system contain serotonin and catecholamines, the shade of their luminescence depends on the quantitative ratio of which. Where these two structures are located side by side, the shade of the glow is approximately the same. Discussion. It is assumed that the implementation of a complex response to the primary chemoreceptor information on the composition of the chyme, obtained by the fluorescent cells of the diffuse endocrine system of the major duodenal papilla, occurs through the exchange of biogenic amines between them and the adrenergic nerve structures of the duodenal papilla.

Use of Continuous Electrical Impedance Measurement for Accurate Nerve Block in Rabbits.

To perform an effective and safe nerve block, the needle must be placed near the target nerve while avoiding nerve damage. Our objective was to conduct an animal study to determine whether changes in electrical impedance (EI) could be used to guide the needle and achieve a safe and accurate nerve block. We measured the EI of rabbit tissues during ultrasound-guided sciatic nerve block using a bipolar needle via the in-plane needle approach. The EI values and needle track on the ultrasound monitor were video-recorded. When there was a change in the EI, the needle advancement was stopped, and a stained anesthetic was injected. Subsequently, the animals were euthanized, and the anesthetic-stained tissue was examined via dissection, while the other tissue was preserved at -80°C for microscopic analysis. The EI remained stable as the needle advanced through the muscle (extraneural); however, it markedly decreased when the needle tip contacted the nerve or slightly punctured the epineurium (paraneural). The mean extra- and paraneural EIs were 4.92 ± 1.31 kΩ (range, 2.39-9.67 kΩ) and 2.86 ± 0.96 kΩ (range, 1.66-5.13 kΩ), respectively. Examination of the dissections and cryostat sections showed anesthetic delivery around the nerve. EI values differed between extra- and paraneural sites, and monitoring these values allowed prediction of the needle tip location with respect to the target nerve. Real-time EI measurement could improve the nerve block.

Three-dimensional histology vs. serial section histology in the treatment of primary basal cell carcinoma: a randomized, prospective, blinded study of 569 tumours.

For basal cell carcinoma (BCC), only few controlled data have been published so far, which directly compare micrographically controlled surgery with conventional serial section histology. In addition to Mohs surgery, which uses cryostat sections, also three-dimensional histology (3D-histology), based on paraffin sections, is available to ensure complete control of the margins and basic sections. To investigate the rate of local recurrence (LR) as well as the number of required re-excisions for basal cell carcinomas with serial section histology vs. 3D-histology. We compared serial sections histology with 3D-histology in a prospective, randomized, controlled blinded trial and analysed 569 BCC of all subtypes up to 30mm diameter, 287 BCC in the 3D group and 282 BCC in the serial section group. Excisions were performed with adapted primary resection margin according to location and size of the tumour. Surgeons were blinded at the time of surgery as they did not know which histological method will be used. Both methods used paraffin sections. Both groups did not differ regarding patients age, tumour location, tumour diameter, tumour subtypes or primary resection margins. In the serial section group, re-excisions were required in 21%; 24 tumours (8.4%) recurred after a median of 2.2years. In the 3D-histology group, re-excisions were required in 39%; 10 tumours recurred (3.5%) after a median of 2.8years. The recurrence rates differed significantly between both groups. Mean follow-up was 4.5years. 3D-histology is a useful technique to detect tumour outgrowths at the excision margins, but required a high rate of re-excisions. 3D-histology was associated with a significantly lower LR rate than serial section histology.

Acute Invasive Fungal Sinusitis: A 30-Year Review of Pathology Practice and Possible Utility of the DiffQuik® Stain.

Acute invasive fungal rhinosinusitis (AIFRS) is a fulminant disease with a high mortality rate. Here, we review the clinical and pathologic features of AIFRS over a 30-year period at a tertiary-care academic center focusing on diagnostic practice, especially the use of intra-operative consultation (IOC). A 1-year trial of intraoperative staining with DiffQuik® (DQ) was also assessed. There were 202 cases from 104 patients with AIFRS. The incidence of AIFRS increased over time (6.8 per year in the past decade versus 1.9 before 2009) as did the number of IOCs per case (2.4 per case in the past decade versus 0.6 before 2009). Disagreement between final diagnosis and IOC was seen in 8.3 % of patients, and the block-by-block error rate was 14.9 %. Fusarium was the most common fungus identified. An attempt to categorize the fungal organism based on histopathology was performed in 85.6 % of patients, with 91 % agreement with microbial cultures or PCR. Fungal hyphae were subjectively easier to identify on DQ stained cryostat sections compared to on routine hematoxylin and eosin stained sections and hyphae were identified in all 5 blocks in which organisms were present. The increasing incidence of AIFRS and expanding use of IOC indicate a need to improve and standardize the diagnostic protocol. The use of DQ as visual aid in IOC for AIFRS may be useful.

Histoarchitectural and Histoenzymatic Studies on Gizzard of Guinea Fowl (Numida meleagris)

Background: The gizzard is a muscular stomach of the Gastrointestinal tract of bird that holds sparing crystals which aids as the mechanical multure component for food material to be ingested by birds. Its specialty is to grinding of ingested food material after secretion of HCL (Hydrochloric Acid) and pepsinogen enzymes in the proventriculus. Methods: The twelve samples of gizzard of guinea fowl were fixed in 10% NBF (Neutral buffered formalin) and Bouin’s fluid for histological while chilled acetone and chilled ethanol for histochemical studies. The fresh unfixed tissues were used for the cryostat section for the demonstration of different enzymes.Result: The tubular glands were main feature of mucosal layer but sometime acinar glands also found as the replacement of tubular glands with lymphoid aggregation. The horizontal koilin was also noticed between long mucosal folds. Tunica submucosa was discovered in squeezed manner in comparison to the mucosa. The tunica muscularis has shown inner circular and outer longitudinal as the thick layer. It was intermingling with collagen and elastic fibers. Histochemically the gizzard was examined for glycogen, alkaline phosphatase, acid phosphatase and succinic dehydrogenase.

Optimized Immunostaining of Embryonic and Early Postnatal Mouse Brain Sections.

The mammalian neocortex, the outer layer of the cerebrum and most recently evolved brain region, is characterized by its unique areal and laminar organization. Distinct cortical layers and areas can be identified by the protein expression of graded transcription factors and molecular determinants that define the identity of different projection neurons. Thus, specific detection and visualization of protein expression is crucial for assessing the identity of neocortical neurons and, more broadly, for understanding early and late developmental mechanisms and function of this complex system. Several immunostaining/immunofluorescence methods exist to detect protein expression. Published protocols vary with regard to subtle details, which may impact the final outcome of the immunofluorescence. Here, we provide a detailed protocol, suitable for both thin cryostat sections and thick vibratome sections, which has successfully worked for a wide range of antibodies directed against key molecular players of neocortical development. Ranging from early technical steps of brains collection down to image analysis and statistics, we include every detail concerning sample inclusion and sectioning, slide storage and optimal antibody dilutions aimed at reducing non-specific background. Routinely used in the lab, our background-optimized immunostaining protocol allows efficient detection of area- and layer- specific molecular determinants of distinct neocortical projection neurons. Graphic abstract: Workflow chart for the optimized immunostaining protocol of mouse brain sections. A. A flow chart for different steps of the optimized immunostaining protocol on both thin cryostat and thick vibratome sections. B. Example for immunostaining against Satb2 and Ctip2 on a thin coronal section (20 μm) at the level of the somatosensory cortex. The first column to the left shows the binning system where 6 bins can be overlaid on the image. On the bottom, an example of counting analysis showing the percentage of marker-positive cells normalized to the total number of DAPI or Hoechst-positive cells. C. Example for immunostaining against Satb2 and Ctip2 on a GFP+ thick vibratome section (200 μm). Images are taken at low magnification (10x, left) and high magnification (40x, right). The graph shows a counting of the percentage of Ctip2-positive neurons normalized to the total number of GFP-electroporated neurons on high-magnification images. Images on B and C are modified from Harb et al. (2016).

Immunofluorescence of GFAP and TNF-α in the Mouse Hypothalamus.

Immunofluorescence is a reliable method for identifying specific proteins in neuronal and glial cell populations of the hypothalamus. Several immunofluorescence protocols are available to detect protein markers and neuropeptides in the hypothalamus; however, published methods may vary in subtle details that can potentially impact the final outcome of the procedure. Here, we provide a detailed protocol suitable for thin cryostat sections, which has been successful for specific antibodies directed against key markers of hypothalamic neurons and glial cells. We include every detail concerning brain tissue collection, processing, sectioning, and labeling with optimal dilutions of antibodies with the aim of reducing non-specific background. Our background-optimized immunostaining protocol has been routinely used in the lab and allows efficient detection of specific neuropeptides, glial cells, and markers of inflammation and endoplasmic reticulum stress in the hypothalamus.

Descriptive analysis of Fibulin-3 and the extracellular vesicle marker, Alix, in drusen from a small cohort of postmortem human eyes

Fibulin-3 (Fib3) is a secreted glycoprotein that is expressed in the retina and has been associated with drusen formation in age-related macular degeneration (AMD). The purpose of this study was to assess whether Fib3 is associated with extracellular vesicles (EVs) in drusen from non-diseased and AMD human donors. De-identified sections of human eyes were received from the National Disease Research Institute (NDRI, Philadelphia). Donor eyes were either non-diseased (no known ocular pathology) or had been diagnosed with AMD. Retinal cryostat sections were labeled with primary antibodies targeted to Fib3, Apolipoprotein E (ApoE; a drusen marker), and ALG-2 interacting protein X (Alix, an EV marker) for confocal imaging (Leica TCS SP8). Fib3-positive (Fib3+) puncta were detected on the apical region of the RPE layer and within large AMD drusen. Alix-positive (Alix+) puncta were also detected in a single AMD druse, where a number were Fib3+ and the remaining were Fib3-negative. Similarly, there were Fib3+ puncta that were Alix-negative. Fib3 and Alix also showed a degree of colocalization in the photoreceptor outer segments of the neural retina. Our data suggest that the Alix+ puncta are EV-rich populations that accumulate, together with Fib3, within the drusen matrix during AMD. The EV population is likely heterogeneous, such that there are sub-populations with different cargo content.

Diagnostic utility of imprint cytology of ovarian neoplasm: A cyto-histological comparison

Background: Intra-operative cytology (IOC) is a method of intra-operative pathological evaluation of cytology smears. The most important indication for IOC is to establish or confirm diagnosis rapidly. Other advantages of IOC are its simple, inexpensive, excellent preservation of cellular details, with no loss of tissues as occurs with cryostat sections and adequacy of surgical margins. Frozen sections are well established method for providing rapid and accurate intra-operative diagnosis. Objective: To evaluate the diagnostic utility of imprint cytology and assess the accuracy of imprint cytology with histopathological diagnosis obtained from ovarian neoplasm. Materials and Methods: Imprint smears were prepared immediately from all the ovariotomy specimens. The specimens were sent for histopathology along with prepared smears to categorize as benign, borderline and malignant. Sensitivity, specificity and accuracy were assessed. Results : Among the 99 specimens studied, 80 were benign, 9 borderline and 10 malignancy according to histopathology. We found that sensitivity, specificity, for benign lesions were 100%, 42.1% and for malignant lesions were 80%, 91.9% respectively. The overall accuracy was 89%. Conclusion : Imprint smear of ovarian neoplasm gives a rapid diagnosis with 89% accuracy against histopathology with added advantages. Imprint cytology is an adjunct to frozen section for providing an intra-operative diagnosis; however, in resource poor settings, it can be used as a stand-alone method for aiding intra-operative decision making. Keywords: Imprint smear, Histopathology, Ovarian neoplasm, Intraoperative cytology.

The Discovery of the Hepatitis D Virus: Three Princes of Serendip and the Recognition of Autoantibodies to Liver-Kidney Microsomes.

The Discovery of the Hepatitis D Virus: Three Princes of Serendip and the Recognition of Autoantibodies to Liver-Kidney Microsomes.

Determination of the Involvement of Autologous Mesenchymal Bone Marrow Stem Cells in Reparative Chondrogenesis

Summary. Articular cartilage injuries are observed in 60% of arthroscopies. Degenerative changes of the articular cartilage (osteoarthritis) is the most common disease of all the joints, which affects at least 10-13% of the population over the age of 60 years. Significant progress has been made in the treatment of pain and inflammation, but the evidence base for improving the articular cartilage repair remains weak. Despite the seemingly positive results of a number of studies on the use of mesenchymal stem cells (MSCs) for the articular cartilage restoration, the question of the nature of their participation in this process remains controversial. Objective. The objective of the study was to determine the involvement of autologous mesenchymal bone marrow stem cells in reparative chondrogenesis. Materials and Methods. Experimental and morphological determination of the participation of undifferentiated autologous bone marrow MSCs in reparative chondrogenesis was carried out on fifteen rabbits. MSCs of bone marrow marked with a red fluorescent dye PKH-26 (Sigma, USA) was injected into the cavity of the injured joint on the 3rd day after the articular cartilage defect formation. The presence of the transplanted labeled autologous MSCs was examined in these animals on cryostat sections on the seventh, fourteenth and twenty-first days after implantation with fluorescence microscopy. The characteristic red glow was observed in case of the presence of labeled cells in the regenerated cartilage tissue. Results. The presence of transplanted labeled autologous MSCs on cryostat sections was determined in all studied animals. On the 7th day, the cells were arranged diffusely, without noticeable accumulations. On the 14th day, the labeled cells fluoresced in the red zone of the spectrum and created compact conglomerates mainly at the defect zone. On the 21th day, the labeled cells were found only in the regenerate zone. The data obtained indicate the direct participation of autologous MSCs in the processes of chondroreparation after the exogenous implantation into the joint cavity. Conclusions. The method of autologous bone marrow MSCs labeling using PKH-26 fluorescent dyes has shown that autologous bone marrow MSCs are directly involved in the processes of chondroreparation when they are injected intraarticularly.

Retina Organoid Transplants Develop Photoreceptors and Improve Visual Function in RCS Rats With RPE Dysfunction.

PurposeTo study if human embryonic stem cell–derived photoreceptors could survive and function without the support of retinal pigment epithelium (RPE) after transplantation into Royal College of Surgeons rats, a rat model of retinal degeneration caused by RPE dysfunction.MethodsCSC14 human embryonic stem cells were differentiated into primordial eye structures called retinal organoids. Retinal organoids were analyzed by quantitative PCR and immunofluorescence and compared with human fetal retina. Retinal organoid sheets (30–70 day of differentiation) were transplanted into immunodeficient RCS rats, aged 44 to 56 days. The development of transplant organoids in vivo in relation to the host was examined by optical coherence tomography. Visual function was assessed by optokinetic testing, electroretinogram, and superior colliculus electrophysiologic recording. Cryostat sections were analyzed for various retinal, synaptic, and donor markers.ResultsRetinal organoids showed similar gene expression to human fetal retina transplanted rats demonstrated significant improvement in visual function compared with RCS nonsurgery and sham surgery controls by ERGs at 2 months after surgery (but not later), optokinetic testing (up to 6 months after surgery) and electrophysiologic superior colliculus recordings (6–8 months after surgery). The transplanted organoids survived more than 7 months; developed photoreceptors with inner and outer segments, and other retinal cells; and were well-integrated within the host.ConclusionsThis study, to our knowledge, is the first to show that transplanted photoreceptors survive and function even with host's dysfunctional RPE. Our findings suggest that transplantation of organoid sheets from stem cells may be a promising approach/therapeutic for blinding diseases.

Pathological identification of brain tumors based on the characteristics of molecular fragments generated by laser ablation combined with a spiking neural network.

Quick and accurate diagnosis helps shorten intraoperative waiting time and make a correct plan for the brain tumor resection. The common cryostat section method costs more than 10 minutes and the diagnostic accuracy depends on the sliced and frozen process and the experience of the pathologist. We propose the use of molecular fragment spectra (MFS) in laser-induced breakdown spectroscopy (LIBS) to identify different brain tumors. Formation mechanisms of MFS detected from brain tumors could be generalized into 3 categories, for instance, combination, reorganization and break. Four kinds of brain tumors (glioma, meningioma, hemangiopericytoma, and craniopharyngioma) from different patients were used as investigated samples. The spiking neural network (SNN) classifier was proposed to combine with the MFS (MFS-SNN) for the identification of brain tumors. SNN performed better than conventional machine learning methods for the analysis of similar and limited MFS information. With the ratio data type, the identification accuracy achieved 88.62% in 2 seconds.

Test-retest reproducibility of atomic force microscopy measurements of human trabecular meshwork stiffness

Purpose: The purpose of the present study was to quantify test-retest reproducibility of measurements of stiffness of the human trabecular meshwork (HTM) by atomic force microscopy (AFM).
 Methods: Eleven 40 μm radial limbal cryostat sections from a fresh human donor rim were mounted on charged slides and rehydrated at room temperature. Stiffness at four TM locations (anterior to posterior along Schlemm’s canal) was measured by AFM. At each location, a 6 x 6 grid was sampled. Indentation points were evenly distributed over a 20 μm x 20 μm area, with a rate of one load/unload cycle per second. Measurements were then repeated for calculation of test-retest variability.
 Results: The test-retest coefficients of variation for the four measurement locations (anterior to posterior) were 24.39, 25.28, 12.74, and 14.26%, respectively, with a notable drop in the two posterior locations compared to the anterior. The test-retest coefficient for the sections was 19.17%. For the entire eye, the test-retest coefficient of variation for the measurement of the TM stiffness was 17.13%. Young’s moduli consistently decreased from anterior to posterior location.
 Conclusions: Wide regional variation suggests that single value does little to fully describe the complex array of TM stiffness levels within the eye, and future studies of TM stiffness assessed by AFM should include multiple tissue samples from each eye, with documentation of the anterior-posterior location of each measurement.

Optimal cutting temperature medium embedding and cryostat sectioning are valid for cardiac myofilament function assessment.

To maximize data obtainment from valuable cardiac tissue, we hypothesized that myocardium fixed in optimal cutting temperature (OCT) medium for histology could also be used to investigate the function of myofilament proteins in situ. We compared tissue prepared via conventional liquid nitrogen (LN) snap freezing with tissue fixed in OCT and then sectioned in fiber-parallel orientation. We found that actin-myosin Ca2+ sensitivity, activation rate by Ca2+, cooperativity along the thin filament, as well as cross-bridge cycling rate were unaffected by OCT storage and could reliably be interpreted after sectioning. Absolute values in maximum force generation per cross-sectional area, as well as passive strain, are difficult to investigate after sectioning, as myofibrillar continuity along the preparation cannot be guaranteed. We have shown that myocardial tissue stored in OCT and sectioned before analysis is available for functional analysis, a valuable means of maximizing usage of precious cardiac biopsies.NEW & NOTEWORTHY Myocardial tissue in optimal cutting temperature (OCT) fixation and cryostat sectioning was tested as a means of storing and preparing tissue for myofilament function analysis in relation to conventional liquid nitrogen freezing and dissection. Actomyosin interaction, Ca2+ force activation, and passive compliance were tested. The study concluded that OCT storage and cryostat sectioning do not interfere with the actomyosin cross-bridge dynamics or Ca2+ activation but that absolute tension values suffer and may not be investigated by this method.

Phasor approach to autofluorescence lifetime imaging FLIM can be a quantitative biomarker of chronic renal parenchymal injury

Diabetic kidney disease continues to be the leading cause of chronic kidney disease, often advancing to end stage kidney disease. In addition to the well characterized glomerular alterations including mesangial expansion, podocyte injury, and glomerulosclerosis, tubulointerstitial fibrosis is also an important component of diabetic kidney injury. Similarly, tubulointerstitial fibrosis is a critical component of any chronic kidney injury. Therefore, sensitive and quantitative identification of tubulointerstitial fibrosis is critical for the assessment of long-term prognosis of kidney disease. Here, we employed phasor approach to fluorescence lifetime imaging, commonly known as FLIM, to understand tissue heterogeneity and calculate changes in the tissue autofluorescence lifetime signatures due to diabetic kidney disease. FLIM imaging was performed on cryostat sections of snap-frozen biopsy material of patients with diabetic nephropathy. There was an overall increase in phase lifetime (τphase) with increased disease severity. Multicomponent phasor analysis shows the distinctive differences between the different disease states. Thus, phasor autofluorescence lifetime imaging, which does not involve any staining, can be used to understand and evaluate the severity of kidney disease.

Changes in Vascular Smooth Muscle and Collagen Content in Placental Vessels in Preeclampsia [04L

INTRODUCTION: Preeclampsia results in part from abnormal myometrial invasion by placental spiral arteries. It is unknown if and how fetal vessels remodel to compensate for these changes. We sought to characterize preeclampsia-associated vascular remodeling by comparing vessels within villi of placentas collected from preeclamptic and normotensive patients. METHODS: Carilion Clinic IRB approval was obtained prior to patient enrollment. Singleton pregnancies complicated by preeclampsia and normotensive controls were identified on Labor and Delivery. Placental biopsies were collected following delivery. Smooth muscle (alpha-SMA) and Type III collagen (COLIII) were identified by immunostaining of cryostat sections. Confocal microscopy was used to measure areas of alpha-SMA and COLIII positivity surrounding vessels. Ratios of alpha-SMA or COLIII positivity to vessel area were calculated. These ratios, which represent relative smooth muscle and collagen content, were compared between vessels from preeclamptic and normotensive placentas. RESULTS: Measurements from 342 vessels from 10 placentas (from five preeclamptic patients and five controls) were included in this analysis. Compared to vessels within placentas of normotensive patients, placental vessels of preeclamptic patients demonstrated a significantly reduced area of alpha-SMA positivity (2.48 vs 3.97; P=.008). A trend was seen towards decreased collagen surrounding vessels in preeclamptic placentas, though this was not statistically significant (4.33 vs 5.66; P=.054). CONCLUSION: Preeclampsia is associated with a decrease in vascular smooth muscle within placental villi and may be associated with decreased collagen content. This may represent a mechanism to increase transplacental flow of oxygen in the setting of impaired spiral artery remodeling, but further studies are needed to clarify this relationship.

Organic secretion in ductal cells of the sublingual salivary gland of ferret: Histochemical observations

The presence of organic secretion in ductal cells of the sublingual salivary gland of ferret has been questioned, which prompted the present investigation. Paraffin or cryostat sections from aldehyde fixed or quenched sublingual glands of this species were tested for some amino acid residues, mucosubstances, oxidative and hydrolytic enzymes, and lectins. The glands showed inconspicuous ducts of simple appearances on routine histology. The histochemical procedures, however, revealed a granulated substance in the apical (periluminal) region of ductal cells, which contained tryptophan, disulphides, neutral mucosubstances, αFuc and GalNAc, and showed chloroacetate esterase activity. Occurrence of the substance varied between different ducts of the same gland and/or cells of the same duct. The ductal cells also showed diffuse peroxidase and acid phosphatase, and Golgi-like thiamine pyrophosphatase activities. Acetylcholinesterase-positive nerve fibres embraced the ducts. The results support a particular localisation of protein-bound amino acid residues and enzymatic catalytic activities indicative of organic secretion, possibly tissue kallikrein, in sublingual ductal cells of ferret.

Optimal Cutting Temperature Medium‐Embedding Is a Valid Method for Storing and Preparing Myocardial Biopsies Preceding Myofilament Function‐Assessment

Background and objectiveOptimal Cutting Temperature (OCT) media is commonly used for histological characterisation. Myocardial tissues from clinical biopsies are precious and thus utilising them for both histological and functional assessment is an attractive prospect. This study was designed to assess whether OCT media stored samples can be used for preparing myocardial tissue for myofilament function‐assessment.MethodsLiquid nitrogen (LN) – and OCT‐stored myocardium from mouse was compared using a permeabilised fibre set‐up for functional characterisation of thin and thick filament‐interaction including Ca2+ sensitivity (pCa50), cooperativity along the thin filament (nH), cross‐bridge cycling rate (ktr), Ca2+ activated maximum tension (Fmax)/cross sectional area, cross‐bridge recruitment upon Ca2+ activation, as well as passive strain (kstrain) and the relative titin contribution to passive stiffness. LN samples were prepared in a conventional fashion and OCT‐stored tissue was first sectioned in parallel with papillary muscle longitudinal axis before chemical demembranisation and further trimming under the microscope.ResultsCa2+ sensitivity (pCa50): LN (n=8): 5.75 ±0.019, OCT (n=8): 5.69 ± 0.022 SEM (p>0.05). Cooperativity along the thin filament (nH): LN (n=8): 2.63 ± 0.24, OCT (n=8): 2.53 ± 0.25 SEM (p>0.05). Fmax: LN (n=8): 19.6 ± 1.9, OCT (n=8): 11.6 ± 1.7 mN/mm2 SEM (p<0.05). Ktr: LN (n=8): 37.5±1.6, OCT (n=8): 37.9 ± 1.6 s−1 SEM (p<0.05). Relative cross‐bridge recruitment upon full Ca2+ activation: LN (n=8): 0.32 ± 0.015, OCT (n=7): 0.31 ± 0.014 SEM (p<0.05). kstrain: LN (n=7): 13.9 ± 0.67, OCT (n=7): 11.4 ± 0.49 L−1, p<0.01). Relative titin contribution to stiffness at 20 % stretch from slack: LN (n=6): 0.52 ± 0.07, OCT (n=7): 0.80 ± 0.03 SEM, (p<0.01).ConclusionsOCT‐storage and cryostat sectioning before mounting fibre bundles on a force‐measuring rig can be used for investigating relative measures of function depending on active force development, ie pCa‐tension relationship, cooperativity along the thin filament and cross‐bridge cycling rate. There appears to be no negative effect on the amount of cross‐bridge recruitment upon Ca2+ activation compared to a conventionally LN‐stored and prepared sample. Absolute values such as Fmax, as well as passive strain, need to be interpreted with great caution as they clearly differ between mode of preparation and thus these parameters are not suited for investigation. We speculate this may be due to how cutting of fibre bundles using a cryostat, to an unknown degree, disrespects the natural continuity of the bundles and thus hampers force relay.Support or Funding InformationThis work was supported by: Swedish Stately Grant for Medical Research (ALF‐means) & Lars Hierta Memorial Fund