Induced Tissue Changes Research Articles

Radiation-induced eye impairments

The problem of the impact of ionizing radiation on eye structures is one of the most important and topical in radiobiology. Knowledge of tissue radiosensitivity and induced tissue changes in the eye is essential for understanding the nature of radiation damage and the behavior of medical professionals. In this publication, the early and late determined radiation-induced impairments of the eye are presented as a result of radiotherapy or radiation incidents. The risk factors determining post-irradiation eye reactions, pathophysiological mechanisms, clinical picture, diagnosis, prevention, and new approaches in therapy are presented. We found that the typically found radiation-induced eye impairments are those of the eyelids – dermatosis and madarosis, conjunctiva – acute conjunctivitis, varied damage to the lacrimal apparatus, cornea – edema and ulceration, iris – rarely inflammation or neovascularization, sclera – rarely atrophy or necrosis, eye lens – cataract, retina – characteristic retinopathy, and optical nerve – rarely neuropathy. We provide the current consensus on prevention and treatment protocols in radiation-induced eye impairments, though most suggested treatment so far should qualify as experimental.

P15.14.A DIFFERENTIATE TREATMENT EFFECTS FROM DISEASE PROGRESSION IN TREATED BRAIN TUMOR: PROMISING RESULTS USING DELAYED CONTRAST MRI

Abstract BACKGROUND Treatment effects are reported in about 30% of treated glioma and brain metastases (BMs) characterized by increasing volume of contrast enhancing regions on MRI, similarly to tumor progression. Data interpretation are often difficult. The most methodology used to distinguish this 2 conditions is perfusion MRI; treatment effects are characterized by a decreased relative cerebral blood volume (rCBV) while persistent or progressive tumors by high rCBV. Limits of perfusion MRI are represented by overlap between two conditions, low spatial resolution, and presence of susceptibility artifacts. To overcome this crucial issue, encouraging results have been obtained using delayed contrast MRI that through contrast clearance analysis (CCA) allows to calculate high resolution maps called “treatment response assessment maps” (TRAMs). We prospectively evaluated patients with doubtful framework between treatment effects (TE) and tumor progression (TP) aiming to assess the ability of CCA in differentiate this two conditions in a cohort of treated brain tumor patients. MATERIAL AND METHODS Maps (TRAMS) were calculated through contrast clearance analysis (CCA) by subtracting ceT1MRI images acquired 5 minutes after contrast injection from images acquired at least 60-105 minutes after. Diagnosis of persistent tumors or disease progression was based on presence of contrast clearance (CC) region in blue, and treatment effects on contrast accumulation (CA) region in red. Data of CCA were compared to perfusion MRI. All images were uploaded, co-registered and elaborate into the image workstation of Brainlab. Whole tumor and enhancing tumor volumes were segmented and perfusion measures in area of signal alteration. TRAMs were generated and at last the calculation of effective volume of viable tumor tissue (CC region) and treatment-induced tissue changes within each lesion (CA region) have been calculated. RESULTS From February 2021 and November 2022, 62 patient (96 delayed-contrast-MRI) with doubtful framework between TE and TP underwent CCA and perfusion-MRI. Twenty-five treated for newly diagnosed glioma, 13 for recurrence, and 37 for BMs. Significantly increased rCBV value in CC regions compared to CA was observed (p=0.01). Patients with TP showed significantly greater CC that subjects with stable disease/partial response 33% vs 78% (p=0.01). TRAMs metric were in agreement with perfusion results. Greater accuracy of TRAMs compared to perfusion-MRI in discriminate TE from TP 81% vs 69% has been observed. CONCLUSION In our experience CCA tool compared to perfusion MRI had a greater accuracy in discriminating true tumor progression from treatment induced tissue changes, and can be of improvement in patient management.

Two-photon fluorescence lifetime imaging microscopy of NADH metabolism in HIV-1 infected cells and tissues.

Rapid detection of microbial-induced cellular changes during the course of an infection is critical to understanding pathogenesis and immunological homeostasis. In the last two decades, fluorescence imaging has received significant attention for its ability to help characterize microbial induced cellular and tissue changes in in vitro and in vivo settings. However, most of these methods rely on the covalent conjugation of large exogenous probes and detection methods based on intensity-based imaging. Here, we report a quantitative, intrinsic, label-free, and minimally invasive method based on two-photon fluorescence lifetime (FLT) imaging microscopy (2p-FLIM) for imaging 1,4-dihydro-nicotinamide adenine dinucleotide (NADH) metabolism of virally infected cells and tissue sections. To better understand virally induced cellular and tissue changes in metabolism we have used 2p-FLIM to study differences in NADH intensity and fluorescence lifetimes in HIV-1 infected cells and tissues. Differences in NADH fluorescence lifetimes are associated with cellular changes in metabolism and changes in cellular metabolism are associated with HIV-1 infection. NADH is a critical co-enzyme and redox regulator and an essential biomarker in the metabolic processes. Label-free 2p-FLIM application and detection of NADH fluorescence using viral infection systems are in their infancy. In this study, the application of the 2p-FLIM assay and quantitative analyses of HIV-1 infected cells and tissue sections reveal increased fluorescence lifetime and higher enzyme-bound NADH fraction suggesting oxidative phosphorylation (OxPhos) compared to uninfected cells and tissues. 2p-FLIM measurements improve signal to background, fluorescence specificity, provide spatial and temporal resolution of intracellular structures, and thus, are suitable for quantitative studies of cellular functions and tissue morphology. Furthermore, 2p-FLIM allows distinguishing free and bound populations of NADH by their different fluorescence lifetimes within single infected cells. Accordingly, NADH fluorescence measurements of individual single cells should provide necessary insight into the heterogeneity of metabolic activity of infected cells. Implementing 2p-FLIM to viral infection systems measuring NADH fluorescence at the single or subcellular level within a tissue can provide visual evidence, localization, and information in a real-time diagnostic or therapeutic metabolic workflow.

Monitoring radiotherapy induced tissue changes in localized prostate cancer by multi-parametric magnetic resonance imaging (MP-MRI)

PurposeTo evaluate the utility of multi-parametric magnetic resonance imaging (MP-MRI), including dynamic contrast-enhanced MRI and diffusion-weighted MRI, for monitoring tumor tissue changes after volumetric-modulated arc radiotherapy in localized prostate cancer (PCa), and to compare the radiotherapy induced tumor tissue changes between conventional, moderate and extreme hypofractionated groups. Furthermore, we aimed to evaluate if follow-up by MRI has an incremental value compared to the standard care by prostate-specific antigen (PSA) serum level measurement. Materials and methodsFifty-five men (mean age: 70±5 [SD] years; range: 60–79 years) with biopsy-proven PCa underwent MRI examination before radiotherapy, and at 3 and 12 months after radiotherapy. Pharmacokinetic analysis post-processing platform with dedicated software (Tissue 4D) was used to generate colorized parametric maps of enhancing tumors. The volume transfer constant (Ktrans), reflux constant (Kep), and initial area under curve (iAUC) were calculated from the tumors. Tumor apparent diffusion coefficient (ADC) value was measured on the ADC map. The patients were allocated into three radiotherapy groups: 17 conventional (39×2Gy), 16 moderate (20×3Gy) and 22 extreme hypofractionated (5×7.25Gy) regimen. ResultsSixty lesions were detected in the prostates of the 55 patients. Follow-up MRI showed decreases in tumor size and degree of enhancement. Ktrans, Kep, and iAUC all decreased at 3 months (P<0.001, respectively) and decreased further at 12 months (P<0.001, respectively). ADC increased at 3 months (P<0.001) and increased further at 12 months (P<0.001). There were no significant differences in the percentage changes of the measured MP-MRI parameters of the tumors from baseline to 12 months between the conventional, moderate and extreme hypofractionated regimen groups. ConclusionMP-MRI is a reliable tool for lesion detection and follow-up, providing both qualitative and quantitative data.

Effects of formalin fixation and temperature on MR relaxation times in the human brain.

Post-mortem MRI of the brain is increasingly applied in neuroscience for a better understanding of the contrast mechanisms of disease induced tissue changes. However, the influence of chemical processes caused by formalin fixation and differences in temperature may hamper the comparability with results from in vivo MRI. In this study we investigated how formalin fixation and temperature affect T1, T2 and T2* relaxation times of brain tissue. Fixation effects were examined with respect to changes in water content and crosslinking. Relaxometry was performed in brain slices from five deceased subjects at different temperatures. All measurements were repeated after 190 days of formaldehyde immersion. The water content of unfixed and fixed tissue was determined using the wet-to-dry ratio following drying. Protein weight was determined with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Fixation caused a strong decrease of all relaxation times, the strongest effect being seen on T1, with a reduction of up to 76%. The temperature coefficient of T1 was lower in the fixed than unfixed tissue, which was in contrast to T2, where an increase of the temperature coefficient was observed following fixation. The reduction of the water content after fixation was in the range of 1-6% and thus not sufficient to explain the changes in relaxation time. Results from SDS-PAGE indicated a strong increase of the protein size above 260 kDa in all brain structures examined. Our results suggest that crosslinking induced changes of the macromolecular matrix are responsible for T1 shortening and a decreased temperature dependency. The relaxation times provided in this work should allow optimization of post-mortem MRI protocols for the brain.

Review of temperature dependence of thermal properties, dielectric properties, and perfusion of biological tissues at hyperthermic and ablation temperatures.

The application of supraphysiological temperatures (>40°C) to biological tissues causes changes at the molecular, cellular, and structural level, with corresponding changes in tissue function and in thermal, mechanical and dielectric tissue properties. This is particularly relevant for image-guided thermal treatments (e.g. hyperthermia and thermal ablation) delivering heat via focused ultrasound (FUS), radiofrequency (RF), microwave (MW), or laser energy; temperature induced changes in tissue properties are of relevance in relation to predicting tissue temperature profile, monitoring during treatment, and evaluation of treatment results. This paper presents a literature survey of temperature dependence of electrical (electrical conductivity, resistivity, permittivity) and thermal tissue properties (thermal conductivity, specific heat, diffusivity). Data of soft tissues (liver, prostate, muscle, kidney, uterus, collagen, myocardium and spleen) for temperatures between 5 to 90°C, and dielectric properties in the frequency range between 460 kHz and 3 GHz are reported. Furthermore, perfusion changes in tumors including carcinomas, sarcomas, rhabdomyosarcoma, adenocarcinoma and ependymoblastoma in response to hyperthmic temperatures up to 46°C are presented. Where appropriate, mathematical models to describe temperature dependence of properties are presented. The presented data is valuable for mathematical models that predict tissue temperature during thermal therapies (e.g. hyperthermia or thermal ablation), as well as for applications related to prediction and monitoring of temperature induced tissue changes.

Postherpetic Neuralgia

Postherpetic neuralgia (PHN) is a neuropathic sequelae in 8% to 27% of individuals with prior varicella zoster virus infection and herpes zoster resulting in retrograde demyelination, neurotoxic reactive oxygen species levels, and proinflammatory cytokine activation of microglia. Pain management strategies are well documented, but not always effective. Laser therapy has shown utility in nerve injury-related pain disorders and was considered a potentially efficacious intervention. Case report. Class 4 therapeutic laser treatment was applied with a dual wavelength GaAlAs (810 nm), GaAl (980 nm) laser, 2 to 4 W, 50% duty cycle, 10 Hz pulse active phase, 2.5 cm diameter aperture, scanning technique with skin contact, 10-minute treatment, 600 to 1200 J total, energy density of 3.5 to 7.1 J/cm average per session, and power density from 0.41 to 0.82 W/cm for 8 treatments. Outcome measures included the Neuropathy Pain Scale Questionnaire as the primary outcome measure, with the Numeric Pain Scale and total area of allodynia touch sensitivity as secondary outcome measurements. The author reports a case of PHN of 15-year duration resistant to prior interventions. Weekly laser therapy treatment over 8 weeks resulted in reduced 0 to 10 Numeric Pain Scale score from 8 to 0, Neuropathy Pain Scale Questionnaire total score from 39 to 4, and allodynia over a 60 cm surface area of the upper trunk and posterior arm totally resolved, with resolution continued at 14-month follow-up. Theoretically, laser therapy induced tissue changes in this case occurring at and below the skin surface altering inflammatory and excitatory peripheral mechanisms noted to take place in the PHN patient. Peripheral nociceptor firing must be brought back to normal thresholds to resolve such chronic neuropathic pain and inhibit the possible central sensitization component. Anti-inflammatory cytokines, growth factors, nitric oxide, adenosine triphosphate (ATP), and other mechanisms stimulated by laser therapy as noted in medical literature may be central to the favorable response seen in this patient. Controlled clinical trials of class 4 laser therapy in the PHN patient population with similar doses would be beneficial to determine if this is an effective treatment option in PHN.

Imaging thermal expansion and retinal tissue changes during photocoagulation by high speed OCT

Visualizing retinal photocoagulation by real-time OCT measurements may considerably improve the understanding of thermally induced tissue changes and might enable a better reproducibility of the ocular laser treatment. High speed Doppler OCT with 860 frames per second imaged tissue changes in the fundus of enucleated porcine eyes during laser irradiation. Tissue motion, measured by Doppler OCT with nanometer resolution, was correlated with the temperature increase, which was measured non-invasively by optoacoustics. In enucleated eyes, the increase of the OCT signal near the retinal pigment epithelium (RPE) corresponded well to the macroscopically visible whitening of the tissue. At low irradiance, Doppler OCT revealed additionally a reversible thermal expansion of the retina. At higher irradiance additional movement due to irreversible tissue changes was observed. Measurements of the tissue expansion were also possible in vivo in a rabbit with submicrometer resolution when global tissue motion was compensated. Doppler OCT may be used for spatially resolved measurements of retinal temperature increases and thermally induced tissue changes. It can play an important role in understanding the mechanisms of photocoagulation and, eventually, lead to new strategies for retinal laser treatments.

Comparison of Intraprostatic Ethanol Diffusion Using a Microporous Hollow Fiber Catheter Versus a Standard Needle

Transurethral intraprostatic ethanol chemoablation of the prostate has shown promising preliminary clinical results for benign prostatic hyperplasia with some variability in clinical outcome. This is likely due to the uneven prostate diffusion caused by varying resistance of the tissue type in which the tip of the needle is embedded. We examined whether the distribution of the injectable in the canine prostate could be improved using a microporous hollow fiber catheter (Twin Star Medical, Minneapolis, Minnesota). The prostate was exposed in 9 mongrel dogs. A single injection of 98% ethanol was delivered in each lobe using a microporous hollow fiber catheter and a standard needle. Prostates were harvested and fixed in 10% formalin. After injection 2.5 mm step sections were obtained and scanned. The ethanol induced tissue lesions were traced on hematoxylin and eosin sections. Three-dimensional reconstructions were created and the volume of each prostate lesion was calculated using stereology. Ethanol induced tissue changes were seen bilaterally in 8 of 9 ethanol injected prostates. In all cases the lesion created by microporous hollow fiber catheter injection was larger than that in the contralateral lobe injected with the control needle. When data were pooled, the hollow fiber catheter injection produced significantly greater tissue changes than the control needle injection (p = 0.03). Improved distribution and absent backflow were seen when using the microporous hollow fiber catheter, supporting its potential as a new method to treat prostate disease.

Two-Photon Microscopy for Non-Invasive, Quantitative Monitoring of Stem Cell Differentiation

BackgroundThe engineering of functional tissues is a complex multi-stage process, the success of which depends on the careful control of culture conditions and ultimately tissue maturation. To enable the efficient optimization of tissue development protocols, techniques suitable for monitoring the effects of added stimuli and induced tissue changes are needed.Methodology/Principal FindingsHere, we present the quantitative use of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) as a noninvasive means to monitor the differentiation of human mesenchymal stem cells (hMSCs) using entirely endogenous sources of contrast. We demonstrate that the individual fluorescence contribution from the intrinsic cellular fluorophores NAD(P)H, flavoproteins and lipofuscin can be extracted from TPEF images and monitored dynamically from the same cell population over time. Using the redox ratio, calculated from the contributions of NAD(P)H and flavoproteins, we identify distinct patterns in the evolution of the metabolic activity of hMSCs maintained in either propagation, osteogenic or adipogenic differentiation media. The differentiation of these cells is mirrored by changes in cell morphology apparent in high resolution TPEF images and by the detection of collagen production via SHG imaging. Finally, we find dramatic increases in lipofuscin levels in hMSCs maintained at 20% oxygen vs. those in 5% oxygen, establishing the use of this chromophore as a potential biomarker for oxidative stress.Conclusions/SignificanceIn this study we demonstrate that it is possible to monitor the metabolic activity, morphology, ECM production and oxidative stress of hMSCs in a non-invasive manner. This is accomplished using generally available multiphoton microscopy equipment and simple data analysis techniques, such that the method can widely adopted by laboratories with a diversity of comparable equipment. This method therefore represents a powerful tool, which enables researchers to monitor engineered tissues and optimize culture conditions in a near real time manner.

Full‐Motion Two‐ and Three‐Dimensional Pulmonary Vein Imaging by Intracardiac Echocardiography After Pulmonary Vein Isolation

The pulmonary veins (PVs) are topographically complex and motile, so angiographic visualization of the PVs anatomy is limited. An imaging technique that accurately portrays the pulmonary vein (PV) anatomy would be valuable during and after catheter ablation procedures. We investigated whether three-dimensional (3D) intracardiac echocardiography (ICE) can visualize radiofrequency (RF)-induced tissue changes after PV isolation. We performed 3D ICE studies with a 9F, 9-MHz ICE catheter after segmental or extended PV isolation. The ICE catheter was placed 3-4 cm inside the PV ostium and mounted onto a pullback device. Sequential two-dimensional (2D) images of the full length of the vein were obtained in 0.3 mm steps with cardiac and respiratory cycle gating. Each image was fed into a computer, and the aggregate data set was reconstructed into a 3D, full-motion image. RF lesion location and lesion size were studied on 67 pullback images from 29 patients. The 2D and 3D reconstruction was possible for 27 left superior PVs, 13 left inferior PVs, 26 right superior PVs, and one right inferior PV. The ablation site was identified 3-7 mm inside the PV ostium, and a 1/2 - 4/5 circumferential area was ablated with no clinically relevant stenosis. No significant differences were found on the ablated area or ablation site between segmental and extensive PV isolation. The 2D and 3D ICE of the PVs provides detailed anatomical information of the proximal PVs, and RF-induced tissue changes in the PV wall can be visualized by ICE.

Skin tissue water and laser Doppler blood flow during a menstrual cycle

Previous work offers conflicting evidence concerning whether basal skin blood flow (SBF) varies with the menstrual cycle. Our goal was to determine the extent of cycle-dependent changes in forearm SBF parameters and if they are linked to local tissue water content and arm volume changes. Both arms of 10 premenopausal women were evaluated three times during their cycle (days 4, 12 and 22) and 10 postmenopausal women were evaluated once. Each evaluation included laser Doppler blood flow parameters (perfusion, volume and velocity), skin temperature, arm volume and local tissue water determined by a new tissue dielectric constant method. Estradiol and progesterone concentrations were determined at each visit by using saliva samples and analysis showed the expected pattern of variation over the cycle. Main results showed no significant cycle-dependent variations in any SBF parameter, temperature, tissue water or arm volume. Postmenopausal women had significantly less estradiol levels, but did not differ with respect to premenopausal women in any measured parameter except for a slightly less blood velocity (P<0.01). We hypothesize that that the absence of changes in tissue water and arm volume in our group may account for the constancy of the measured SBF parameters. However, in other populations, hormonally induced tissue changes may occur and contribute to the cycle-dependent changes in resting blood perfusion that have been reported. This possibility suggests that it may be useful to measure and report a skin tissue water index when serial changes in blood perfusion are being investigated.

MRI investigation of the threshold for thermally induced blood-brain barrier disruption and brain tissue damage in the rabbit brain.

The ability of MRI-derived thermometry to predict thermally induced tissue changes in the brain was tested, and the thermal thresholds for blood-brain barrier (BBB) disruption and brain tissue damage were estimated. In addition, the ability of standard MRI to detect threshold-level effects was confirmed. These safety thresholds are being investigated to provide guidelines for clinical thermal ablation studies in the brain. MRI-monitored focused ultrasound heating was delivered to 63 locations in 26 rabbits. Tissue changes were detected in T(2)-weighted imaging and T(1)-weighted imaging (with and without contrast) and with light microscopy. The probability for tissue damage as a function of the accumulated thermal dose, the peak temperature achieved, the applied acoustic energy, and the peak acoustic power was estimated with probit regression. The discriminative abilities of these parameters were compared using the areas under the receiver operator characteristic (ROC) curves. In MRI, BBB disruption was observed in contrast-enhanced T(1)-weighted imaging shortly after the ultrasound exposures, sometimes accompanied by changes in T(2)-weighted imaging. Two days later, changes in T(2)-weighted imaging were observed, sometimes accompanied by changes in T(1)-weighted imaging. In histology, tissue damage was seen at every location where MRI changes were observed, ranging from small (diameter <1.0 mm) areas of tissue necrosis to severe vascular damage and associated hemorrhagic infarct. In one location, small (diameter: 0.8 mm) damage was not detected in MRI. The thermal dose and peak temperature thresholds were between 12.3-40.1 equivalent min at 43 degrees C and 48.0-50.8 degrees C, respectively, and values of 17.5 equivalent min at 43 degrees C and 48.4 degrees C were estimated to result in tissue damage with 50% probability. Thermal dose and peak temperature were significantly better predictors than the applied acoustic energy and peak acoustic power (P < 0.01). BBB disruption was always accompanied by tissue damage. The temperature information was better than the applied acoustic power or energy for predicting the damage than the ultrasound parameters. MRI was sensitive in detecting threshold-level damage.

Supramaximal cholecystokinin displaces Munc18c from the pancreatic acinar basal surface, redirecting apical exocytosis to the basal membrane.

Exocytosis at the apical surface of pancreatic acinar cells occurs in the presence of physiological concentrations of cholecystokinin (CCK) but is inhibited at high concentrations. Here we show that Munc18c is localized predominantly to the basal membranes of acinar cells. Supramaximal but not submaximal CCK stimulation caused Munc18c to dissociate from the plasma membrane, and this displacement was blocked by protein kinase C (PKC) inhibitors. Conversely, whereas the CCK analog CCK-OPE alone failed to displace Munc18c from the membrane, this agent caused Munc18c displacement following minimal PKC activation. To determine the physiological significance of this displacement, we used the fluorescent dye FM1-43 to visualize individual exocytosis events in real-time from rat acinar cells in culture. We showed that supramaximal CCK inhibition of secretion resulted from impaired apical secretion and a redirection of exocytic events to restricted basal membrane sites. In contrast, CCK-OPE evoked apical exocytosis and could only induce basolateral exocytosis following activation of PKC. Infusion of supraphysiological concentrations of CCK in rats, a treatment that induced tissue changes reminiscent of mild acute pancreatitis, likewise resulted in rapid displacement of Munc18c from the basal membrane in vivo.