Retro is the new modern: Contemporary application of gold impregnation staining on brain cryosections for digital image analysis

Background and purpose: Since the times of Golgi and Cajal, impregnation with gold or silver has been used to visualize microscopic details of the nervous tissue. Although immunohistochemistry has largely replaced impregnation techniques, they are still used, and there is a growing interest in combining them with modern image analysis methods for quantitative studies in neuroscience. The aim of this research was to modify the gold chloride impregnation method published by Schmued to improve consistency of staining, to be adequate for digital image analysis. Materials and methods: Brains of 8 six-month-old female Wistar rats were fixed in 4% PFA, cryoprotected in sucrose and flash‑frozen in liquid nitrogen. Neighboring sets of coronal sections were chosen for gold impregnation, Nissl staining and MAP2 immunohistochemistry. Whole-slide images and images of specific regions were taken for analysis. Results: Myelinated fibers were stained dark reddish to brown on goldstained sections, and other tissue was yellowish, which gave an excellent contrast for digital image analysis. Gold staining was consistent in all regions, and no major artifacts were noticed. When compared to Nissl and MAP2, only myelinated structures were stained with gold impregnation. Conclusions: Modified gold impregnation method is an alternative that’s on par with traditional myelin staining methods. The new, modified gold impregnation method gives a consistent and reproducible staining suitable for digital image analysis. It can be useful in morphometric evaluation of nervous tissue and investigation of neuropathological changes in nervous tissue, especially for quantitative studies

The influence of PROGINS alu insertion on the function of progesterone receptors and the modulation of premature delivery risk

HIPOTEZA: PROGINS alu insercija u genu za progesteronski receptor predstavlja genetički rizični čimbenik za prijevremeni porod. CILJEVI ISTRAŽIVANJA: Ciljevi ovog istraživanja bili su ispitati povezanost genske mutacije u progesteronskom receptoru (PROGINS alu insercija) i pojave spontanih prijevremenih poroda i utvrditi je li navedena mutacija genetički rizični čimbenik za prijevremeni porod. USTROJ STUDIJE: Retrospektivno istraživanje parova. ISPITANICI I METODE: Ispitivana populacija u ovom istraživanju su rodilje s Klinike za ginekologiju i obstetriciju KBC Osijek. Ispitivana populacija trudnica koje su rodile prijevremeno sastoji se od 100 uzoraka (50 rodilje, 50 novorođenčad). Kontrolnu skupinu (50 rodilje, 50 novorođenčad) čine terminske trudnice kojima je porod krenuo spontano. Kriterij za isključivanje su jatrogeni prijevremeni porod (indukcije radi preeklampsije, dijabetesa i drugo). Uzorkovala se krv trudnica jednokratno venepunkcijom, a krv novorođenčadi dobivena je iz pupčane vrpce. DNA je iozolirana iz pune krvi komercijalnim spin kolonama te primjenom PCR-a i elektroforeze ispitano postojanje PROGINS alu insercije. Rezultati su statistički analizirani. REZULTATI: Svakom uzorku dodijeljen je odgovarajući genotip. Nakon statističke analize uočeno je da nema značajne razlike u učestalosti PROGINS alu insercije između ispitanika i kontrolne skupine. Usporedba rodilja iz kontrolne skupine i rodilja ispitanica (p = 0.8456); usporedba djece iz kontrolne skupina i djece ispitanika ( p = 0.558). Korišteni su Hi kvadrat test, Student t-test te Mann-Whitney U test za nezavisne uzorke. Razina značajnosti postavljena je na p = 0,05. ZAKLJUČAK: Iz navedenih rezultata istraživanja možemo zaključiti da PROGINS alu insercija u genu za progesteronski receptor ne predstavlja rizični čimbenik za prijevremeni porod.HYPOTHESIS: PROGINS alu insertion in the progesterone receptor gene is a genetic risk factor for preterm delivery. OBJECTIVES: The objectives of this study were to investigate the association of gene mutation in the progesterone receptor (PROGINS alu insertion) with the occurrence of spontaneous preterm birth and to determine whether the mutation is a genetic risk factor for preterm delivery. STUDY DESIGN: Retrospective study. PARTICIPANTS AND METHODS: The study population in this study is a maternity ward from the Clinic for Gynecology and Obstetrics, Clinical Hospital Center Osijek. The study population of pregnant women who gave birth prematurely consists of 100 samples (50 women giving birth, 50 newborns). The control group (50 women giving birth, 50 newborns) consists of term pregnant women who have given birth spontaneously. Exclusion criteria are iatrogenic preterm delivery (induction for preeclampsia, diabetes and more). The blood of pregnant women was sampled by venipuncture, and the blood of newborns was obtained from the umbilical cord. DNA was isolated from whole blood by commercial spin columns and the existence of PROGINS alu insertion was examined using PCR and electrophoresis. The results were statistically analyzed. RESULTS: Each sample was assigned a corresponding genotype. After the statistical analysis, it was found that there was no significant difference in the frequency of PROGINS alu insertion between the subjects and the control group.Comparison of maternity control and respondent groups (p = 0.8456); comparison of children from the control group and children of the subjects (p = 0.558). Tests that were used are Chi-squared test, Student t-test and Mann-Whitney U test for independent samples. The significance level was set at p = 0.05. CONCLUSION: From the aforementioned research results we can conclude that PROGINS alu insertion in the progesterone receptor gene is not a genetic risk factor for preterm delivery

The effect of hop extract, alendronate and their combination on the expression of interneuronal immunohistochemical markers and nerve fiber morphology in the brain of ovariectomized Wistar rats

Cilj istraživanja: Ispitati utjecaj ekstrakta hmelja, alendronata i njihove kombinacije na izražaj imunohistokemijskih biljega interneurona u mozgu štakora, morfologiju živčanih vlakana mozga štakora i izražaj mikrotubula u aksonima mozga štakora. Ustroj studije: Studija je ustrojena kao randomizirani kontrolirani pokus u kojem je tkivo mozga ovarijektomiranih ženki štakora tretiranih alendronatom i/ili ekstraktom hmelja uspoređivano s tkivom mozga netretiranih ovarijektomiranih ženki štakora i zdravih netretiranih ženki štakora. Ispitanici i metode: U istraživanje su uključena tkiva mozgova 50 ženki Wistar štakora podijeljenih u 5 skupina, ovisno o terapiji koju su primili i o tome jesu li ovarijektomirani ili ne. Korištena je impregnacijska metoda zlatom za prikaz živčanih vlakan i imunohistokemijsko bojenje, pomoću kojeg su na tkivu mozga prikazani epitopi za MAP2, kalbindin, kalretinin i parvalbumin. Rezultati su analizirani računalnim programom ImageJ te su statistički obrađeni programom Statistica. Rezultati: Tretmani ekstraktom hmelja i/ili alendronatom imali su utjecaj na izražaj imunohistokemijskih biljega interneurona, morfologiju živčanih vlakana i izražaj mikrotubula u aksonima u pojedinim regijama mozga ovarijektomiranih Wistar štakora. Zaključak: Primjena ekstrakta hmelja i/ili alendronata utječe na izražaj imunohistokemijskih biljega interneurona, na morfologiju živčanih vlakana i izražaj mikrotubula u mozgu ovarijektomiranih štakora.Objectives: To examine the influence of hop extract, alendronate and their combination on the expression of interneuron immunohistochemical markers in rat brain, morphology of rat brain nerve fibers and expression of microtubules in rat brain axons. Study design: The study was designed as a randomized controlled trial in which brain tissue of ovariectomized female rats treated with alendronate and/or hop extract was compared to brain tissues of untreated ovariectomized female rats and with healthy, untreated female rats. Materials and methods: The study included brain tissues of 50 female Wistar rats divided into 5 groups, depending on the therapy they received and whether they were ovariectomized or not. An impregnation technique of gold chloride staining was used to display nerve fibers. MAP2, calbindin, calretinin and parvalbumin were shown using immunohistochemistry. The results were analyzed with ImageJ software and statistically processed with Statistica software. Results: Treatments with hop extract and/or alendronate affected the expression of immunohistochemical markers of interneurons, the morphology of nerve fibers and the expression of microtubules in axons in certain brain regions of ovariectomized Wistar rats. Conclusion: Treatments with hop extract and/or alendronate affects the expression of immunohistochemical markers of interneurons, morphology of nerve fibers and expression of microtubules in the brain of ovariectomized rats

The effect of hop extract, alendronate and their combination on the expression of interneuronal immunohistochemical markers and nerve fiber morphology in the brain of ovariectomized Wistar rats

Cilj istraživanja: Ispitati utjecaj ekstrakta hmelja, alendronata i njihove kombinacije na izražaj imunohistokemijskih biljega interneurona u mozgu štakora, morfologiju živčanih vlakana mozga štakora i izražaj mikrotubula u aksonima mozga štakora. Ustroj studije: Studija je ustrojena kao randomizirani kontrolirani pokus u kojem je tkivo mozga ovarijektomiranih ženki štakora tretiranih alendronatom i/ili ekstraktom hmelja uspoređivano s tkivom mozga netretiranih ovarijektomiranih ženki štakora i zdravih netretiranih ženki štakora. Ispitanici i metode: U istraživanje su uključena tkiva mozgova 50 ženki Wistar štakora podijeljenih u 5 skupina, ovisno o terapiji koju su primili i o tome jesu li ovarijektomirani ili ne. Korištena je impregnacijska metoda zlatom za prikaz živčanih vlakan i imunohistokemijsko bojenje, pomoću kojeg su na tkivu mozga prikazani epitopi za MAP2, kalbindin, kalretinin i parvalbumin. Rezultati su analizirani računalnim programom ImageJ te su statistički obrađeni programom Statistica. Rezultati: Tretmani ekstraktom hmelja i/ili alendronatom imali su utjecaj na izražaj imunohistokemijskih biljega interneurona, morfologiju živčanih vlakana i izražaj mikrotubula u aksonima u pojedinim regijama mozga ovarijektomiranih Wistar štakora. Zaključak: Primjena ekstrakta hmelja i/ili alendronata utječe na izražaj imunohistokemijskih biljega interneurona, na morfologiju živčanih vlakana i izražaj mikrotubula u mozgu ovarijektomiranih štakora.Objectives: To examine the influence of hop extract, alendronate and their combination on the expression of interneuron immunohistochemical markers in rat brain, morphology of rat brain nerve fibers and expression of microtubules in rat brain axons. Study design: The study was designed as a randomized controlled trial in which brain tissue of ovariectomized female rats treated with alendronate and/or hop extract was compared to brain tissues of untreated ovariectomized female rats and with healthy, untreated female rats. Materials and methods: The study included brain tissues of 50 female Wistar rats divided into 5 groups, depending on the therapy they received and whether they were ovariectomized or not. An impregnation technique of gold chloride staining was used to display nerve fibers. MAP2, calbindin, calretinin and parvalbumin were shown using immunohistochemistry. The results were analyzed with ImageJ software and statistically processed with Statistica software. Results: Treatments with hop extract and/or alendronate affected the expression of immunohistochemical markers of interneurons, the morphology of nerve fibers and the expression of microtubules in axons in certain brain regions of ovariectomized Wistar rats. Conclusion: Treatments with hop extract and/or alendronate affects the expression of immunohistochemical markers of interneurons, morphology of nerve fibers and expression of microtubules in the brain of ovariectomized rats

The influence of PROGINS alu insertion on the function of progesterone receptors and the modulation of premature delivery risk

HIPOTEZA: PROGINS alu insercija u genu za progesteronski receptor predstavlja genetički rizični čimbenik za prijevremeni porod. CILJEVI ISTRAŽIVANJA: Ciljevi ovog istraživanja bili su ispitati povezanost genske mutacije u progesteronskom receptoru (PROGINS alu insercija) i pojave spontanih prijevremenih poroda i utvrditi je li navedena mutacija genetički rizični čimbenik za prijevremeni porod. USTROJ STUDIJE: Retrospektivno istraživanje parova. ISPITANICI I METODE: Ispitivana populacija u ovom istraživanju su rodilje s Klinike za ginekologiju i obstetriciju KBC Osijek. Ispitivana populacija trudnica koje su rodile prijevremeno sastoji se od 100 uzoraka (50 rodilje, 50 novorođenčad). Kontrolnu skupinu (50 rodilje, 50 novorođenčad) čine terminske trudnice kojima je porod krenuo spontano. Kriterij za isključivanje su jatrogeni prijevremeni porod (indukcije radi preeklampsije, dijabetesa i drugo). Uzorkovala se krv trudnica jednokratno venepunkcijom, a krv novorođenčadi dobivena je iz pupčane vrpce. DNA je iozolirana iz pune krvi komercijalnim spin kolonama te primjenom PCR-a i elektroforeze ispitano postojanje PROGINS alu insercije. Rezultati su statistički analizirani. REZULTATI: Svakom uzorku dodijeljen je odgovarajući genotip. Nakon statističke analize uočeno je da nema značajne razlike u učestalosti PROGINS alu insercije između ispitanika i kontrolne skupine. Usporedba rodilja iz kontrolne skupine i rodilja ispitanica (p = 0.8456); usporedba djece iz kontrolne skupina i djece ispitanika ( p = 0.558). Korišteni su Hi kvadrat test, Student t-test te Mann-Whitney U test za nezavisne uzorke. Razina značajnosti postavljena je na p = 0,05. ZAKLJUČAK: Iz navedenih rezultata istraživanja možemo zaključiti da PROGINS alu insercija u genu za progesteronski receptor ne predstavlja rizični čimbenik za prijevremeni porod.HYPOTHESIS: PROGINS alu insertion in the progesterone receptor gene is a genetic risk factor for preterm delivery. OBJECTIVES: The objectives of this study were to investigate the association of gene mutation in the progesterone receptor (PROGINS alu insertion) with the occurrence of spontaneous preterm birth and to determine whether the mutation is a genetic risk factor for preterm delivery. STUDY DESIGN: Retrospective study. PARTICIPANTS AND METHODS: The study population in this study is a maternity ward from the Clinic for Gynecology and Obstetrics, Clinical Hospital Center Osijek. The study population of pregnant women who gave birth prematurely consists of 100 samples (50 women giving birth, 50 newborns). The control group (50 women giving birth, 50 newborns) consists of term pregnant women who have given birth spontaneously. Exclusion criteria are iatrogenic preterm delivery (induction for preeclampsia, diabetes and more). The blood of pregnant women was sampled by venipuncture, and the blood of newborns was obtained from the umbilical cord. DNA was isolated from whole blood by commercial spin columns and the existence of PROGINS alu insertion was examined using PCR and electrophoresis. The results were statistically analyzed. RESULTS: Each sample was assigned a corresponding genotype. After the statistical analysis, it was found that there was no significant difference in the frequency of PROGINS alu insertion between the subjects and the control group.Comparison of maternity control and respondent groups (p = 0.8456); comparison of children from the control group and children of the subjects (p = 0.558). Tests that were used are Chi-squared test, Student t-test and Mann-Whitney U test for independent samples. The significance level was set at p = 0.05. CONCLUSION: From the aforementioned research results we can conclude that PROGINS alu insertion in the progesterone receptor gene is not a genetic risk factor for preterm delivery