Raman spectroscopy system for real-time diagnosis of clinically significant prostate cancer tissue.

Prostate cancer (PCa) is a significant healthcare problem worldwide. Current diagnosis and treatment methods are limited by a lack of precise in vivo tissue analysis methods. Real-time cancer identification and grading could dramatically improve current protocols. Here, we report the testing of a thin optical probe using Raman spectroscopy (RS) and classification methods to detect and grade PCa accurately in real-time. We present the first clinical trial on fresh ex vivo biopsy cores from an 84 patient cohort. Findings from 2395 spectra measured on 599 biopsy cores show high accuracy for diagnosing and grading PCa. We can detect clinically significant PCa from benign and clinically insignificant PCa with 90% sensitivity and 80.2% specificity. We also demonstrate the ability to differentiate cancer grades with 90% sensitivity and specificity ≥82.8%. This work demonstrates the utility of RS for real-time PCa detection and grading during routine transrectal biopsy appointments.

Localized bioelectrical conduction block from radiofrequency gastric ablation persists after healing: safety and feasibility in a recovery model.

Gastric ablation has demonstrated potential to induce conduction blocks and correct abnormal electrical activity (i.e., ectopic slow-wave propagation) in acute, intraoperative in vivo studies. This study aimed to evaluate the safety and feasibility of gastric ablation to modulate slow-wave conduction after 2 wk of healing. Chronic in vivo experiments were performed in weaner pigs (n = 6). Animals were randomly divided into two groups: sham-ablation (n = 3, control group; no power delivery, room temperature, 5 s/point) and radiofrequency (RF) ablation (n = 3; temperature-control mode, 65°C, 5 s/point). In the initial surgery, high-resolution serosal electrical mapping (16 × 16 electrodes; 6 × 6 cm) was performed to define the baseline slow-wave activation profile. Ablation (sham/RF) was then performed in the mid-corpus, in a line around the circumferential axis of the stomach, followed by acute postablation mapping. All animals recovered from the procedure, with no sign of perforation or other complications. Two weeks later, intraoperative high-resolution mapping was repeated. High-resolution mapping showed that ablation successfully induced sustained conduction blocks in all cases in the RF-ablation group at both the acute and 2 wk time points, whereas all sham-controls had no conduction block. Histological and immunohistochemical evaluation showed that after 2 wk of healing, the lesions were in the inflammation and early proliferation phase, and interstitial cells of Cajal (ICC) were depleted and/or deformed within the ablation lesions. This safety and feasibility study demonstrates that gastric ablation can safely and effectively induce a sustained localized conduction block in the stomach without disrupting the surrounding slow-wave conduction capability.NEW & NOTEWORTHY Ablation has recently emerged as a tool for modulating gastric electrical activation and may hold interventional potential for disorders of gastric function. However, previous studies have been limited to the acute intraoperative setting. This study now presents the safety of gastric ablation after postsurgical recovery and healing. Localized electrical conduction blocks created by ablation remained after 2 wk of healing, and no perforation or other complications were observed over the postsurgical period.

Gastric ablation as a novel technique for modulating electrical conduction in the in vivo stomach.

Gastric motility is coordinated by underlying bioelectrical "slow wave" activity. Slow wave dysrhythmias are associated with motility disorders, including gastroparesis, offering an underexplored potential therapeutic target. Although ablation is widely used to treat cardiac arrhythmias, this approach has not yet been trialed for gastric electrical abnormalities. We hypothesized that ablation can create localized conduction blocks and modulate slow wave activation. Radiofrequency ablation was performed on the porcine serosa in vivo, encompassing a range of parameters (55-85°C, adjacent points forming a line, 5-10 s/point). High-resolution electrical mapping (16 × 16 electrodes; 6 × 6 cm) was applied to define baseline and acute postablation activation patterns. Tissue damage was evaluated by hematoxylin and eosin and c-Kit stains. Results demonstrated that RF ablation successfully induced complete conduction block and a full thickness lesion in the muscle layer at energy doses of 65-75°C for 5-10 s/point. Gastric ablation may hold therapeutic potential for gastric electrical abnormalities in the future.NEW & NOTEWORTHY This study presents gastric ablation as a new method for modulating slow wave activation and propagation in vivo, by creating localized electrical conduction blocks in the stomach, validated by high-resolution electrical mapping and histological tissue analysis. The results define the effective energy dose range for creating conduction blocks, while maintaining the mucosal and submucosal integrity, and demonstrate the electrophysiological effects of ablation. In future, gastric ablation can now be translated toward disrupting dysrhythmic slow wave activation.

A non-opioid analgesic implant for sustained post-operative intraperitoneal delivery of lidocaine, characterized using an ovine model.

Appropriate management of post-operative pain is an ongoing challenge in surgical practice. At present, systemic opioid administration is routinely used for analgesia in the post-operative setting. However, due to significant adverse effects and potential for misuse, there is a perceived need for the development of alternative, opioid-sparing treatment modalities. Continuous infusion of local anesthetic into the peritoneum after major abdominal surgery reduces pain and opioid consumption, and enhances recovery from surgery. Here we describe a non-opioid, poly(ethylene-co-vinyl-acetate) intraperitoneal implant for the sustained delivery of local anesthetic following major abdominal surgery. A radio-opaque core had the required mechanical strength to facilitate placement and removal procedures. This core was enclosed by an outer shell containing an evenly dispersed local anesthetic, lidocaine. Sustained release of lidocaine was observed in an ovine model over days and the movement modelled between peritoneal fluid and circulating plasma. While desirably high levels of lidocaine were achieved in the peritoneal space these were several orders of magnitude higher than blood levels, which remained well below toxic levels. A pharmacokinetic model is presented that incorporates in vitro release data to describe lidocaine concentrations in both peritoneal and plasma compartments, predicting similar release to that suggested by lidocaine concentrations remaining in the device after 3 and 7 days in situ. Histological analysis revealed similar inflammatory responses following implantation of the co-extruded implant and a commercially used silicone drain after three days. This non-opioid analgesic implant provides sustained release of lidocaine in an ovine model and is suitable for moving onto first in human trials.

Understanding the Lingual Frenulum: Histological Structure, Tissue Composition, and Implications for Tongue Tie Surgery.

Lingual frenotomy has become an increasingly common surgical procedure, performed for a broad range of indications from birth through adulthood. This study utilizes histology to define the structure and tissue composition of the lingual frenulum and floor of mouth (FOM) fascia. En bloc specimens of anterior tongue, lingual frenulum, and FOM tissues were harvested from ten embalmed adult cadavers. An additional three fresh tissue cadaveric specimens were frozen with the tongue supported in an elevated position, to enable harvesting and paraffin embedding of the elevated lingual frenulum as a discrete specimen. All 13 specimens were prepared as ten-micron coronal sections using stains to determine the general morphology of the lingual frenulum, its relationship to neighbouring structures (Mason's Trichrome), presence of elastin fibers (Verhoeff-van Gieson), and collagen typing (Picrosirius Red). Our results have shown a submucosal layer of fascia spanning horizontally across the FOM was present in all specimens, with variability in fascial thickness and histologic composition. This FOM fascia suspends the sublingual glands, vessels, and genioglossus from its deep surface. The elevated lingual frenulum is formed by a central fold of this FOM fascia together with the overlying oral mucosa with variability in fascial thickness and composition. With tongue elevation, the fascia mobilizes to a variable extent into the fold forming the frenulum, providing a structural explanation for the individual variability in lingual frenulum morphology seen in clinical practice.

Expression and steroid hormone regulation of TETs and DNMTs in human endometrium.

DNA methyltransferases (DNMTs) and ten-eleven translocation proteins (TETs) facilitate methylation and hydroxymethylation of DNA, respectively. DNMTs are widely studied with conflicting results on their regulation in the endometrium. While the role of TETs in the endometrium remains relatively unexplored. Deregulated expression of TETs and DNMTs are associated with endometrial pathologies. The aim of this study is to characterize the temporal TET expression in endometrium and to determine the hormonal regulation of TETs in comparison to DNMTs. mRNA expressions were quantified by real-time PCR in endometrial tissues from cycling women and localization was determined by immunohistochemistry. Hormonal regulation was investigated in endometrial epithelial and stromal cell lines following a 24 and 48 h treatment cycle. TET1 and 3 mRNA expressions were significantly upregulated in the mid-secretory phase. TET protein expression was ubiquitous in endometrial epithelium throughout the menstrual cycle except during the late-secretory phase, while stromal staining was scattered. TET1 mRNA was significantly upregulated in response to estrogen in stromal cells. Transcriptions of all three TETs were induced in response to progesterone treatment in epithelial cells. Only DNMT3b in epithelial cells and DNMT1 in stromal cells were significantly upregulated upon 24-h estrogen exposure following a significant decrease of DNMT1 when treated with 24 h of estrogen and progesterone. This study suggests that TETs are expressed in a cell-specific, dynamic manner in the endometrium and are responsive to steroid hormones. Investigating the role of TETs individually and with respect to DNMTs, will help to elucidate gene regulatory mechanisms in endometrial biology and pathologies.

Renal denervation in male rats with heart failure improves ventricular sympathetic nerve innervation and function.

Heart failure is characterized by the loss of sympathetic innervation to the ventricles, contributing to impaired cardiac function and arrhythmogenesis. We hypothesized that renal denervation (RDx) would reverse this loss. Male Wistar rats underwent myocardial infarction (MI) or sham surgery and progressed into heart failure for 4 wk before receiving bilateral RDx or sham RDx. After additional 3 wk, left ventricular (LV) function was assessed, and ventricular sympathetic nerve fiber density was determined via histology. Post-MI heart failure rats displayed significant reductions in ventricular sympathetic innervation and tissue norepinephrine content (nerve fiber density in the LV of MI+sham RDx hearts was 0.31 ± 0.05% vs. 1.00 ± 0.10% in sham MI+sham RDx group, P < 0.05), and RDx significantly increased ventricular sympathetic innervation (0.76 ± 0.14%, P < 0.05) and tissue norepinephrine content. MI was associated with an increase in fibrosis of the noninfarcted ventricular myocardium, which was attenuated by RDx. RDx improved LV ejection fraction and end-systolic and -diastolic areas when compared with pre-RDx levels. This is the first study to show an interaction between renal nerve activity and cardiac sympathetic nerve innervation in heart failure. Our findings show denervating the renal nerves improves cardiac sympathetic innervation and function in the post-MI failing heart.

Augmentation with an ovine forestomach matrix scaffold improves histological outcomes of rotator cuff repair in a rat model.

BACKGROUND: Rotator cuff tears can cause significant pain and functional impairment. Without surgical repair, the rotator cuff has little healing potential, and following surgical repair, they are highly prone to re-rupture. Augmenting such repairs with a biomaterial scaffold has been suggested as a potential solution. Extracellular matrix (ECM)-based scaffolds are the most commonly used rotator cuff augments, although to date, reports on their success are variable. Here, we utilize pre-clinical in vitro and in vivo assays to assess the efficacy of a novel biomaterial scaffold, ovine forestomach extracellular matrix (OFM), in augmenting rotator cuff repair. METHODS: OFM was assessed in vitro for primary tenocyte growth and adherence, and for immunogenicity using an assay of primary human dendritic cell activation. In vivo, using a murine model, supraspinatus tendon repairs were carried out in 34 animals. Augmentation with OFM was compared to sham surgery and unaugmented control. At 6- and 12-week time points, the repairs were analysed biomechanically for strength of repair and histologically for quality of healing. RESULTS: OFM supported tenocyte growth in vitro and did not cause an immunogenic response. Augmentation with OFM improved the quality of healing of the repaired tendon, with no evidence of excessive inflammatory response. However, there was no biomechanical advantage of augmentation. CONCLUSIONS: The ideal rotator cuff tendon augment has not yet been identified or clinically implemented. ECM scaffolds offer a promising solution to a difficult clinical problem. Here, we have shown improved histological healing with OFM augmentation. Identifying materials that offset the poorer mechanical properties of the rotator cuff post-injury/repair and enhance organised tendon healing will be paramount to incorporating augmentation into surgical treatment of the rotator cuff.

Stress-related changes to immune cells in the skin prior to wounding may impair subsequent healing.

Higher psychological stress is associated with slower dermal wound healing, but the immunological mechanisms behind this effect are only partially understood. This paper aims to investigate whether immune cells present in the skin prior to wounding can affect subsequent healing in high-stress and low-stress participants. Two studies are presented in which skin biopsies were analysed using immunohistochemistry for numbers of macrophages and Langerhans cells, and immune cell activation (Study 2 only). Immune cells were related to perceived stress levels and subsequent healing. Study 1 included 19 healthy older adults and showed that higher stress was associated with significantly fewer macrophages in the skin. Study 2 included 22 younger adults and showed that higher stress was associated with significantly lower activation of immune cells in the skin. Furthermore, lower activation of immune cells (as measured by human leukocyte antigen (HLA expression)) and fewer Langerhans cells were associated with slower healing. Together these studies show the first preliminary evidence that the number and activation of immune cells in the skin prior to wounding are affected by stress and can impact healing. Larger studies are needed to confirm these effects.

Post-insertion of poloxamer 188 strengthened liposomal membrane and reduced drug irritancy and in vivo precipitation, superior to PEGylation.

The ultimate aim of this study was to develop asulacrine (ASL)-loaded long-circulating liposomes to prevent phlebitis during intravenous (i.v.) infusion for chemotherapy. Poly(ethylene)glycol (PEG) and poloxamer 188-modified liposomes (ASL-PEGL and ASL-P188L) were developed, and ASL was loaded using a remote loading method facilitated with a low concentration of sulfobutyl ether-ß-cyclodextrin as a drug solubilizer. The liposomes were characterized in terms of morphology, size, release properties and stability. Pharmacokinetics and venous tissue tolerance of the formulations were simultaneously studied in rabbits following one-hour i.v. infusion via the ear vein. The irritancy was assessed using a rat paw-lift/lick model after subplantar injections. High drug loading 9.0% w/w was achieved with no drug leakage found from ASL-PEGL or ASL-P188L suspended in a 5% glucose solution at 30days. However, a rapid release (leakage) from ASL-PEGL was observed when PBS was used as release medium, partially related to the use of cyclodextrin in drug loading. Post-insertion of poloxamer 188 to the liposomes appeared to be able to restore the drug retention possibly by increasing the packing density of phospholipids in the membrane. In rabbits (n=5), ASL-P188L had a prolonged half-life with no drug precipitation or inflammation in the rabbit ear vein in contrast to ASL solution. Following subplantar (footpad) injections in rats ASL solution induced paw-lick/lift responses in all rats whereas ASL-P188L caused no response (n=8). PEGylation showed less benefit possibly due to the drug 'leakage'. In conclusion, drug precipitation in the vein and the drug mild irritancy may both contribute to the occurrence of phlebitis caused by the ASL solution, and could both be prevented by encapsulation of the drug in liposomes. Poloxamer 188 appeared to be able to 'seal' the liposomal membrane and enhance drug retention. The study also highlighted the importance of bio-relevant in vitro release study in formulation screening.

Intramucosal bacterial microcolonies exist in chronic rhinosinusitis without inducing a local immune response.

BACKGROUND: Although chronic rhinosinusitis (CRS) causes very significant morbidity, much about its pathogenesis remains uncertain. Recent studies have identified polymicrobial biofilms on the surface of sinus mucosa and Staphylococcus aureus within the sinus mucosa of patients with CRS, both with and without nasal polyps. The pathogenic implications of intramucosal bacteria in CRS are unknown. This study was designed to determine the prevalence and species of bacterial colonies within the sinus mucosa of adult patients with and without CRS and to describe the relationship of these bacterial colonies to the host immune response. METHODS: Sinus mucosa from patients with and without CRS was examined using Gram and Giemsa staining, immunohistochemistry, bacterial culture, and fluorescence in situ hybridization techniques. RESULTS: Bacterial microcolonies were observed within the mucosa in 14 of 18 patients with CRS. In 10 of these patients colonies were positively identified as S. aureus. Staphylococcal microcolonies were observed at a lower level (1 of 8 patients) in normal sinus mucosa. There was no correlation between detection of S. aureus on the mucosal surface and microcolonization of the mucosa. Surprisingly, there was no evidence of an immune reaction to microcolonies. Indeed, fewer T lymphocytes (p = 0.03) and eosinophils (p = 0.03) were counted immediately surrounding the microcolonies compared with uninfected areas of the same tissue. CONCLUSION: Bacterial microcolonies are prevalent within paranasal sinus mucosa and are commonly S. aureus. These microcolonies do not provoke immune detection and may represent a phenotype that actively evades host immunity. This may underpin the recalcitrance of CRS to antibiotic therapy. These findings challenge classic views of both infection and mucosal immunity in human chronic disease. The presence of intramucosal bacteria in samples of normal sinus mucosa also questions the sensitivity of detecting nasal carriage of pathogens by swabbing the surface of the anterior nares.

Bacterial microcolonies exist within the sphenoid bone in chronic rhinosinusitis and healthy controls.

BACKGROUND: Some patients with chronic rhinosinusitis (CRS) exhibit thickening of the sinus bones that has been termed osteitis. The histopathology and microbiology of these changes have not been fully described. The aim of this study was to look for the presence of bacteria and immune cells within samples of bone from patients with and without CRS and correlate these findings to radiological findings. METHODS: Bone on the anterior face of the sphenoid was examined radiologically and histologically in 8 patients with CRS with nasal polyposis, 8 patients with CRS without polyposis, and 6 control patients with pituitary adenomas and normal sinuses. Bone thickness and density were measured by computed tomography (CT) scanning. Bone samples were collected intraoperatively and 20 tissue sections were analyzed for each patient. Bacteria were identified by Giemsa and Gram stains. Immune cells were identified by conventional histology and immunohistochemistry. RESULTS: Small colonies of bacteria were identified within the bone in 3 of 16 CRS patients and 2 of 6 control subjects (p = 0.6). Isolated immune cells were identified within the bone in 3 of 16 CRS patients and 2 of 6 control subjects (p = 0.6) but both bacteria and immune cells occurred together in only 1 case. The presence of bacteria or immune cells within bone samples did not correlate with either bone thickness or bone density. CONCLUSION: This study describes the presence of bacteria and immune cells within a minority of CRS patients and normal controls. The bacterial microcolonies identified do not appear to be the cause of the bone changes seen in many CRS patients.

Are biofilms associated with an inflammatory response in chronic rhinosinusitis?

BACKGROUND: Bacterial biofilms have been identified on the sinonasal mucosa of patients with chronic rhinosinusitis (CRS) but also on control samples. Their role in the disease pathogenesis is unproven. The objective of this study was to further evaluate the role of biofilms in CRS by assessing whether they are associated with an inflammatory response. METHODS: Mucosal samples were collected from 18 patients with CRS and 7 normal subjects. Bacteria on the mucosal surface were identified by Gram stain. Immune cells were identified by Giemsa stain and immunohistochemistry (IHC). The number of local immune cells was recorded beneath areas of the mucosal surface both colonized with and free from bacteria. RESULTS: In CRS patients, biofilms that were directly opposed to a disrupted epithelial layer were associated with more T lymphocytes (p = 0.01), and more macrophages (p = 0.003) than areas of mucosa without bacteria present. Biofilms associated with but not directly opposed to the epithelium were not associated with raised numbers of immune cells. CONCLUSION: Not all surface bacterial colonies are associated with a particular inflammatory response in CRS. Biofilms adherent to a disrupted epithelial layer are associated with higher numbers of immune cells and therefore appear to have a role in the pathogenesis of CRS.