Predrag Brkic

Application of fractal and grey level co-occurrence matrix analysis in evaluation of brain corpus callosum and cingulum architecture.

This aim of this study was to assess the discriminatory value of fractal and grey level co-occurrence matrix (GLCM) analysis methods in standard microscopy analysis of two histologically similar brain white mass regions that have different nerve fiber orientation. A total of 160 digital micrographs of thionine-stained rat brain white mass were acquired using a Pro-MicroScan DEM-200 instrument. Eighty micrographs from the anterior corpus callosum and eighty from the anterior cingulum areas of the brain were analyzed. The micrographs were evaluated using the National Institutes of Health ImageJ software and its plugins. For each micrograph, seven parameters were calculated: angular second moment, inverse difference moment, GLCM contrast, GLCM correlation, GLCM variance, fractal dimension, and lacunarity. Using the Receiver operating characteristic analysis, the highest discriminatory value was determined for inverse difference moment (IDM) (area under the receiver operating characteristic (ROC) curve equaled 0.925, and for the criterion IDM≤0.610 the sensitivity and specificity were 82.5 and 87.5%, respectively). Most of the other parameters also showed good sensitivity and specificity. The results indicate that GLCM and fractal analysis methods, when applied together in brain histology analysis, are highly capable of discriminating white mass structures that have different axonal orientation.

Discriminatory ability of fractal and grey level co-occurrence matrix methods in structural analysis of hippocampus layers

Fractal and grey level co-occurrence matrix (GLCM) analysis represent two mathematical computer-assisted algorithms that are today thought to be able to accurately detect and quantify changes in tissue architecture during various physiological and pathological processes. However, despite their numerous applications in histology and pathology, their sensitivity, specificity and validity regarding evaluation of brain tissue remain unclear. In this article we present the results indicating that certain parameters of fractal and GLCM analysis have high discriminatory ability in distinguishing two morphologically similar regions of rat hippocampus: stratum lacunosum-moleculare and stratum radiatum. Fractal and GLCM algorithms were performed on a total of 240 thionine-stained hippocampus micrographs of 12 male Wistar albino rats. 120 digital micrographs represented stratum lacunosum-moleculare, and another 120 stratum radiatum. For each image, 7 parameters were calculated: fractal dimension, lacunarity, GLCM angular second moment, GLCM contrast, inverse difference moment, GLCM correlation, and GLCM variance. GLCM variance (VAR) resulted in the largest area under the Receiver operating characteristic (ROC) curve of 0.96, demonstrating an outstanding discriminatory power in analysis of stratum lacunosum-moleculare (average VAR equaled 478.1 ± 179.8) and stratum radiatum (average VAR of 145.9 ± 59.2, p < 0.0001). For the criterion VAR ≤ 227.5, sensitivity and specificity were 90% and 86.7%, respectively. GLCM correlation as a parameter also produced large area under the ROC curve of 0.95. Our results are in accordance with the findings of our previous study regarding brain white mass fractal and textural analysis. GLCM algorithm as an image analysis method has potentially high applicability in structural analysis of brain tissue cytoarcitecture.

Hyperbaric oxygenation improves locomotor ability by enhancing neuroplastic responses after cortical ablation in rats.

Objective: To investigate whether hyperbaric oxygenation (HBO) can improve the recovery of motor functions in rats after suction ablation of the right sensorimotor cortex. Methods: The experimental paradigm implies the following groups: Control animals (C), Control + HBO (CHBO), Sham controls (S), Sham control + HBO (SHBO), Lesion group (L), right sensorimotor cortex was removed by suction, Lesion + HBO (LHBO). Hyperbaric protocol: pressure applied 2.5 atmospheres absolute, for 60 minutes, once a day for 10 days. A beam walking test and grip strength meter were used to evaluate the recovery of motor functions. Expression profiles of growth-associated protein 43 (GAP43) and synaptophysin (SYP) were detected using immunohistochemistry. Results: The LHBO group achieved statistically superior scores in the beam walking test compared to the L group. Additionally, the recovery of muscle strength of the affected hindpaw was significantly enhanced after HBO treatment. Hyperbaric oxygenation induced over-expression of GAP43 and SYP in the neurons surrounding the lesion site. Conclusions: Data presented suggest that hyperbaric oxygen therapy can intensify neuroplastic responses by promoting axonal sprouting and synapse remodelling, which contributes to the recovery of locomotor performances in rats. This provides the perspective for implementation of HBO in clinical strategies for treating traumatic brain injuries.

Repetitive Hyperbaric Oxygenation Attenuates Reactive Astrogliosis and Suppresses Expression of Inflammatory Mediators in the Rat Model of Brain Injury

The exact mechanisms by which treatment with hyperbaric oxygen (HBOT) exerts its beneficial effects on recovery after brain injury are still unrevealed. Therefore, in this study we investigated the influence of repetitive HBOT on the reactive astrogliosis and expression of mediators of inflammation after cortical stab injury (CSI). CSI was performed on male Wistar rats, divided into control, sham, and lesioned groups with appropriate HBO. The HBOT protocol was as follows: 10 minutes of slow compression, 2.5 atmospheres absolute (ATA) for 60 minutes, and 10 minutes of slow decompression, once a day for 10 consecutive days. Data obtained using real-time polymerase chain reaction, Western blot, and immunohistochemical and immunofluorescence analyses revealed that repetitive HBOT applied after the CSI attenuates reactive astrogliosis and glial scarring, and reduces expression of GFAP (glial fibrillary acidic protein), vimentin, and ICAM-1 (intercellular adhesion molecule-1) both at gene and tissue levels. In addition, HBOT prevents expression of CD40 and its ligand CD40L on microglia, neutrophils, cortical neurons, and reactive astrocytes. Accordingly, repetitive HBOT, by prevention of glial scarring and limiting of expression of inflammatory mediators, supports formation of more permissive environment for repair and regeneration.

Hyperbaric oxygenation alters temporal expression pattern of superoxide dismutase 2 after cortical stab injury in rats.

Aim To evaluate the effect of hyperbaric oxygen therapy (HBOT) on superoxide dismutase 2 (SOD2) expression pattern after the cortical stab injury (CSI). Methods CSI was performed on 88 male Wistar rats, divided into control, sham, lesioned, and HBO groups. HBOT protocol was the following: pressure applied was 2.5 absolute atmospheres, for 60 minutes, once a day for consecutive 3 or 10 days.‎ The pattern of SOD2 expression and cellular localization was analyzed using real-time polymerase chain reaction, Western blot, and double-label fluorescence immunohistochemistry. Neurons undergoing degeneration were visualized with Fluoro-Jade®B. Results CSI induced significant transient increase in SOD2 protein levels at day 3 post injury, which was followed by a reduction toward control levels at post-injury day 10. At the same time points, mRNA levels for SOD2 in the injured cortex were down-regulated. Exposure to HBO for 3 days considerably down-regulated SOD2 protein levels in the injured cortex, while after 10 days of HBOT an up-regulation of SOD2 was observed. HBOT significantly increased mRNA levels for SOD2 at both time points compared to the corresponding L group, but they were still lower than in controls. Double immunofluorescence staining revealed that 3 days after CSI, up-regulation of SOD2 was mostly due to an increased expression in reactive astrocytes surrounding the lesion site. HBOT attenuated SOD2 expression both in neuronal and astroglial cells. Fluoro-Jade®B labeling showed that HBOT significantly decreased the number of degenerating neurons in the injured cortex. Conclusion HBOT alters SOD2 protein and mRNA levels after brain injury in a time-dependent manner.

Hyperbaric oxygen and in vitro fertilisation.

In vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) are used to help infertile couple to conceive their own biological child. Success of IVF is associated with several factors, although many important factors are, unfortunately, not known. Known prognostic factors include maternal age, ovarian reserve, past reproductive performance, embryonal culture, endometrial receptivity, embryo transfer and luteal phase support. The therapeutic application of hyperbaric oxygen (HBO) has been known over three decades in different fields of medicine. Our objective was to determine the usefulness of HBO therapy during IVF procedure. While it is difficult to prove that HBO is the crucial factor for the success of this IVF/ICSI procedure, it is important to report that pregnancy occurred.

Modulation of rat synaptosomal ATPases and acetylcholinesterase activities induced by chronic exposure to the static magnetic field

Abstract Purpose: It is considered that exposure to static magnetic fields (SMF) may have both detrimental and therapeutic effect, but the mechanism of SMF influence on the living organisms is not well understood. Since the adenosine triphosphatases (ATPases) and acetylcholinesterase (AChE) are involved in both physiological and pathological processes, the modulation of Na+/K+-ATPase, ecto-ATPases and AChE activities, as well as oxidative stress responses were followed in synaptosomes isolated from rats after chronic exposure toward differently oriented SMF. Material and methods: Wistar albino rats were randomly divided into three experimental groups (six animals per group): Up and Down group - exposed to upward and downward oriented SMF, respectively, and Control group. After 50 days, the rats were sacrificed, and synaptosomes were isolated from the whole rat brain and used for testing the enzyme activities and oxidative stress parameters. Results: Chronic exposure to 1 mT SMF significantly increased ATPases, AChE activities, and malondialdehyde (MDA) level in both exposed groups, compared to control values. The significant decrease in synaptosomal catalase activity (1.48 ± 0.17 U/mg protein) induced by exposure to the downward oriented field, compared to those obtained for Control group (2.60 ± 0.29 U/mg protein), and Up group (2.72 ± 0.21 U/mg protein). Conclusions: It could be concluded that chronic exposure to differently oriented SMF increases ATPases and AChE activities in rat synaptosomes. Since brain ATPases and AChE have important roles in the pathogenesis of several neurological diseases, SMF influence on the activity of these enzymes may have potential therapeutic importance

The effects of hyperbaric oxygen treatment on vigility of spermatozoids: Preliminary report

The aim of this study was to evaluate the effect of hyperbaric oxygen treatment (HBO) to the vigilance of spermatozoids. The sperm samples were obtained from the patients (n=10) with diagnosed oligospermia. The motility of spermatozoids in tested samples was not lower than 30% but it did not exceed the critical proportion of 50%. Three groups of samples were formed, with respectively 10 samples in each group. All of them were exposed to HBO conditions of 2.5 ATA for 90 minutes. Group I was exposed to HBO conditions without any addition of medium. Group II was added 5 ml of EBSS solution. Tyrodes solution in volume of 5 ml was added to group III. The evaluation of functional characteristics of sperm was carried out 30 minutes after completion of HBO treatment. Statistically significant increased in motor activity was registered in three all groups (p<0.05). The biggest increased in vigilance of spermatozoids was found in the samples of the group III (p<0.01), exceeding the critical proportion of 50% in all of the samples. The obtained results suggest that the acute exposure of the sperm samples to HBO has favorable impact to functional capacity of spermatozoids in view of their better motility.