Enikö Račeková

Immunosuppressant FK506: focusing on neuroprotective effects following brain and spinal cord injury.

The secondary damage that follows central nervous system (CNS) injury is a target for neuroprotective agents aimed at tissue and function sparing. FK506, a clinically used immunosuppressant, acts neuroprotectively in rat models of brain and spinal cord injury and ischemia. Evidence of in vivo experimental studies highlights the neuroprotective role of FK506 by its direct impact on various cell populations within the CNS. The participation of FK506 in modulation of post-traumatic inflammatory processes is a further potential aspect involved in CNS neuroprotection. In this review we provide an overview of the current laboratory research focusing on the multiple effects of FK506 on neuroprotection following CNS injury.

Fluoro Jade-B detection of dying cells in the SVZ and RMS of adult rats after bilateral olfactory bulbectomy

SummaryA novel fluorochrome, Fluoro-Jade B, was used to detect dying precursor cells in the subventricular zone (SVZ) and rostral migratory stream (RMS) of adult rats after bilateral olfactory bulbectomy and in control intact rats. The animals in experimental group were left to survive 3 days and from 3 till 16 months after surgical procedure. 1.In the control animals, Fluoro-Jade B positive cells were visible in the SVZ and within the whole extent of the RMS. The number of Fluoro-Jade B positive cells increased in the elbow in comparison to the rest parts of the RMS.2.In the experimental animals surviving either 3 days or from 3 till 16 months after bilateral olfactory bulbectomy, Fluoro-Jade B positive cells displayed the similar pattern of distribution as in the control animals. However, some quantitative differences in the labeled cells number along the rostral migratory pathway appeared.3.The average number of degenerating cells within the control SVZ and RMS was 26.24± 0.686. In bulbectomized animals, regardless of survival time, an insignificant increase of Fluoro-Jade B positive cells number occurred.We can conclude that dying of precursor cells is a physiological process running within the SVZ/RMS in both control and experimental animals. Moreover, this physiological process is not influenced by survival period after bilateral olfactory bulbectomy. Our results demonstrate Fluoro-Jade B as a useful marker of dying cells.

Age-Related Changes of NADPH-Diaphorase Positivity in the Rat Rostral Migratory Stream

SummaryAccumulating evidence confirms that nitric oxide (NO), a versatile diffusible signaling molecule, contributes to controling of adult neurogenesis. We have previously shown the timing of NADPH-diaphorase (NADPH-d) positivity within the rat rostral migratory stream (RMS) during the first postnatal month. The present study was designed to describe further age-related changes of NO presence in this neurogenic region. The presence of NO synthesizing cells in the RMS was shown by NADPH-d histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemistry. The phenotypic identity of nitrergic cells was examined by double labeling with GFAP and NeuN. Systematic qualitative and quantitative analysis of NADPH-d-positive cells was performed in the neonatal (P14), adult(5 months) and aging (20 months) rat RMS.1. Nitrergic cells with different distribution pattern and morphological characteristics were present in the RMS at all ages examined. In neonatal animals, small, moderately stained NADPH-d-positive cells were identified in the RMS vertical arm and in the RMS elbow. In adult and aging rats a few labeled cells could be also detected in the RMS horizontal arm. NADPH-d-positive cells in adult and aging rats were characterized by long varicose processes and displayed dark labeling in comparison to the neonatal group.2. Double immunolabeling has revealed that nNOS-immunoreactivity co-localized with that of NeuN. This indicates that nitrergic cells within the RMS are neurons.3. Quantitative analysis showed that the number of NADPH-d-positive cells increases with advancing age.The presence of NO producing cells in the RMS of neonatal adult and aging rats indicates, that this proliferating and migratory area is under the influence of NO throughout the entire life of the animals.

Ependyma as a Possible Morphological Basis of Ischemic Preconditioning Tolerance in Rat Spinal Cord Ischemia Model: Nestin and Fluoro-Jade B Observations

Abstract1. To test our hypothesis that a transient nonlethal ischemic insult benefits the lumbosacral spinal cord ischemic injury, nestin, the marker of proliferating cells, and Fluoro-Jade B, the marker of degenerating cells, were used in rats. Morphological outcome was evaluated after 12-min ischemia versus 12-min ischemia preconditioned by 3-min ischemic period and 30-min recirculation (IPC), in each group followed by 2, 3, and 4 days of posttreatment survival.2. Twelve-minute ischemia, inducing nestin-positivity in ependyma and reactive astrocytes at the L1−3 spinal cord segments, shows this region as the viable region of spinal cord in all postischemic survival periods. On the other hand, abundance of Fluoro-Jade B-positive cells, distributed throughout the dorsal horn and intermediate zone of L4–S2 segments, points out the most injured spinal cord region by ischemia.3. After the same ischemic insult in IPC rats only a few nestin-positive ependymal cell and reactive astrocytes appeared beside the nestin-positive vessels in the lower lumbar and sacral spinal cord segments of all survival periods. The appearance of nestin-positive cells in the spinal cord segments, which “should have been affected” by ischemia indicates protection of this region by the IPC treatment.4. The number and density evaluation of Fluoro-Jade B fluorescent cells of L4–S2 segments after ischemia and IPC confirmed that degenerating cells were significantly reduced in the IPC rats in all survival periods.5. Our results showing the immunohistochemical response of epemdyma, committed to the presence of viable tissue, indicate that the ependymal cells may contribute to the ischemic resistance in the IPC rats.

The effect of maternal body condition on in vivo production of zygotes and behavior of delivered offspring in mice

This study investigated the effects of maternal body condition on oocyte quality and zygote production. Additionally, we examined the possible consequences on somatic parameters and behavior of naturally delivered offspring. We used an experimental model based on overfeeding of outbred mice during intrauterine and early postnatal development to produce the following four types of females: physiological (7%-8%), slightly increased (8%-11%), highly increased (>11%), and low (<7%) body fat content (Echo Magnetic Resonance Imaging). The fertilized females with slightly increased body fat showed increased numbers of spontaneously ovulated oocytes and an increased fertilization index compared with control animals. On the contrary, mice with slightly and highly increased body fat showed increased numbers of isolated immature oocytes and degenerates. Furthermore, animals with increased body fat had significantly decreased deposits of neutral lipids in the cytoplasm of mature oocytes (Nile red staining) and showed lower reduction in DNA cytosine methylation signal in parental pronuclei (5-methylcytosine immunohistochemistry). The highly increased amount of body fat in mothers was accompanied with lower weights in newborn pups and 5-week-old offspring. We also observed several deviations from normal behavior (open-field test and forced swimming test). The females with low body fat displayed a lower fertilization index, a lower percentage of zygotes at pronuclear stage 4 with demethylated DNA cytosine in parental pronuclei, and lower newborn weights. Although delivered offspring were able to gain normal weight by the fifth week of life, there were several deviations from normal behavior observed. Our results show that periconceptional status of maternal body condition adversely affects the quality of oocytes and might be correlated with significant changes during postnatal offspring development. The data documenting later onset of DNA demethylation in zygotes and decreased amounts of neutral lipids in oocytes suggest that the observed alterations in offspring might originate in modifications established at the earliest stages of conceptus development.

Regional differences of proliferation activity in the spinal cord ependyma of adult rats

Increased proliferation activity in the central canal ependyma of adult rodent spinal cord was described after injury and is thought to participate in recovery processes. Proliferation activity is scarce under physiological conditions, but still could be of importance, as in vitro studies showed that the spinal cord ependyma is an internal source of neural stem cells. Data from these studies indicate that there are regional differences in the distribution of proliferation activity along the rostro-caudal axis. We analyzed the proliferation activities in the ependyma within the entire extent of intact adult rat spinal cord. To identify proliferating cells we performed immunohistochemistry either for cell cycle S-phase marker BrdU or for the nuclear protein Ki-67. BrdU and Ki-67 positive cells were counted on sections selected from four spinal cord regions — cervical, thoracic, lumbar and sacral/coccygeal. Analysis showed that the number of BrdU positive cells within the ependyma was very low in all subdivisions of the spinal cord. Both BrdU and Ki-67 labeling revealed a significantly higher number of proliferating cells in the ependyma of sacrococcygeal part in comparison to all other spinal cord regions, suggesting that the caudal spinal cord might have potentially higher regeneration capacity compared to more rostral parts.

Fos Protein Expression in Sacral Spinal Cord in Relation to Early Phase of Cauda Equina Syndrome in Dogs

Abstract1. The aim of the present study is to map the incipient phase of Fos expression in the sacral spinal cord neuronal pools of multiple cauda equina constrictions canine model.2. Fos-positive neurons were found bilaterally in the lateral portion of superficial dorsal horn layers (Laminae I–III) and along the lateral edge of the dorsal horn accompanied by the lateral collateral pathway, fibers of Lissauers tract, terminating at the sacral parasympathetic nucleus. Similarly, high Fos expression was detected in the ventral portion of the dorsal sacral commissure and in the dorsomedial portion of the anterior horns at S1–S3 segment level. Finally, a clearly expressed Fos-positivity was disclosed bilaterally in the neuropil of the nucleus Y in the anterior horn.3. Data from the present study show that continuous stimulation of the central fibers of sacral dorsal root ganglia neurons, i.e., fibers of sacral primary afferents, unlike those using various stimulations of the peripheral fibres offers an unusual pattern of Fos-like immunoreactivity.

Structural and Ultrastructural Study of Rat Testes Influenced by Electromagnetic Radiation

This study was conducted to investigate the influence of whole-body electromagnetic radiation (EMR) on testicular parenchyma of Wistar rats. Sexually mature rats were subjected to pulsed electromagnetic field at frequency of 2.45 GHz and mean power density 2.8 mW/cm2 by 3-h daily applications for 3 wk. Tissue samples were obtained 3 h after the last irradiation and processed by histological techniques for light and transmission electron microscopy. Testes showed apparent degenerative changes of seminiferous epithelium. The seminiferous tubules were mostly irregular in shape, and seminiferous epithelium contained a number of empty spaces of different size. Subsequently, groups of sloughed epithelial cells were often found inside the lumina of tubules. Except for relatively unchanged Sertoli cells, some locations of basal compartment of seminiferous epithelium contained shriveled Sertoli cells with dark cytoplasm. These areas showed degenerative features including necrotizing and shriveled spermatogonia surrounded by empty irregular spaces, and undulating basement membrane. The intertubular spaces were enlarged but interstitial Leydig cells did not show any marked morphological changes. Evidence demonstrates the adverse effects of EMR on testicular parenchyma in rats.

Astrocytic and Vascular Scaffolding for Neuroblast Migration in the Rostral Migratory Stream

New neurons are continuously being added to the olfactory bulb (OB) of adult rodents that are generated in the subventricular zone (SVZ), distant by a few millimeters. Neuronal precursors have to overcome this long distance without the radial-glial migratory scaffold, in contrast to migration mode during embryonic development. The previous model explains migration of precursors from the SVZ through the rostral migratory stream (RMS) to the OB as a movement of neuroblasts along each other, ensheathed by astroglial tubes. Recent results indicate that blood vessels are suitable candidates for neuronal migration guidance in the RMS. These novel findings have changed the former concept accounting for neuronal precursor migration. The aim of our study was to map a pattern of vascularization in the RMS of adult rats and to investigate mutual relations among blood vessels, neuroblasts and astrocytes in this area. Detailed morphological analysis revealed that blood vessels in the RMS are organized in a specific manner. In most of the RMS extent, blood vessels run parallel to the outline of the migratory pathway. Interestingly, the caudal part of the RMS has a unique vasculature organization in which blood vessels create a spiral-like configuration. Chains of neuroblasts enveloped by astrocytes largely align along blood vessels. The exception is the caudal part of the RMS where neuroblasts do not follow non - parallel blood vessels. Our morphological findings suggest that blood vessels and astrocytes may cooperatively form physical substrate - scaffold for the neuroblasts migration in the RMS of adult rats.

AN OLFACTORY BULBECTOMY MODEL TO STUDY PLASTICITY OF THE NERVOUS SYSTEM IN RAT: A REVIEW FOCUSED ON PUBLISHED DATA FROM THE SLOVAK REPUBLIC

This review surveys the main data published at the Institute of Neurobiology, Slovak Republic, pertaining to both morphological and functional observations in the olfactory bulbectomy model. The review utilized this experimental approach in homo- and heterotopic transplantations, in experiments for studying neurodegenaration/regeneration and phenomena of adult neurogenesis. The results, discussed in context of relevant literary data, indicate that the model of olfactory bulbectomy can be utilized to investigate problems not only related to the olfactory system but it can also address the problems of neuronal development, neuronal plasticity and interaction concerning the nervous system as a whole.