Miloš Stanojlović

Low-Dose Dexamethasone Treatment Promotes the Pro-Survival Signalling Pathway in the Adult Rat Prefrontal Cortex

Synthetic glucocorticoid dexamethasone (DEX), a highly potent anti‐inflammatory and immunosuppressive agent, is widely used in the treatment of brain cancer, as well as for inflammatory and autoimmune diseases. The present study aimed to determine whether low‐dose subchronic DEX treatment (100 μg/kg for eight consecutive days) exerts long‐term effects on apoptosis in the adult rat prefrontal cortex (PFC) by examining the expression of cell death‐promoting molecules [poly(ADP‐ribose) polymerase (PARP), p53, procaspase 3, cleaved caspase 3, Bax] and cell‐survival molecules (AKT, Bcl‐2). The results obtained revealed that body, thymus and adrenal gland weights, as well corticosterone levels, in the serum and PFC were reduced 1 day after the last DEX injection. In the PFC, DEX caused activation of AKT, augmentation of pro‐survival Bcl‐2 protein and an enhanced Bcl‐2/Bax protein ratio, as well Bcl‐2 translocation to the mitochondria. An unaltered profile with respect to the protein expression of apoptotic molecules PARP, procaspase 3 and Bax was detected, whereas p53 protein was decreased. Reverse transcriptase ‐polymerase chain reaction analysis showed a decrease of p53 mRNA levels and no significant difference in Bcl‐2 and Bax mRNA expression in DEX‐treated rats. Finally, a DNA fragmentation assay and Fluoro‐Jade staining demonstrated no considerable changes in apoptosis in the rat PFC. Our findings support the concept that low‐dose DEX creates a hypocorticoid state in the brain and also indicate that subchronic DEX treatment activates the pro‐survival signalling pathway but does not change apoptotic markers in the rat PFC. This mechanism might be relevant for the DEX‐induced apoptosis resistance observed during and after chemotherapy of patients with brain tumours.

Repeated low-dose 17β-estradiol treatment prevents activation of apoptotic signaling both in the synaptosomal and cellular fraction in rat prefrontal cortex following cerebral ischemia

Disturbance in blood circulation is associated with numerous pathological conditions characterized by cognitive decline and neurodegeneration. Activation of pro-apoptotic signaling previously detected in the synaptosomal fraction may underlie neurodegeneration in the prefrontal cortex of rats submitted to permanent bilateral common carotid arteries occlusion (two-vessel occlusion, 2VO). 17β-Estradiol (E) exerts potent neuroprotective effects in the brain affecting, among other, ischemia-induced pathological changes. As most significant changes in rats submitted to 2VO were observed on 7th day following the insult, of interest was to examine whether 7 day treatment with low dose of E (33.3 µg/kg/day) prevents formerly reported neurodegeneration and may represent additional therapy during the early post-ischemic period. Role of E treatment on apoptotic pathway was monitored on Bcl-2 family members, cytochrome c, caspase 3 and PARP protein level in the synaptosomal (P2) fraction of the prefrontal cortex. Furthermore, changes of these proteins were examined in the cytosolic, mitochondrial and nuclear fraction, with the emphasis on potential involvement of extracellular signal-regulated kinases (ERK) and protein kinase B (Akt) activation and their role in nuclear translocation of transcriptional nuclear factor kappa B (NF-kB) associated with alteration of Bax and Bcl-2 gene expression. The extent of cellular damage was determined using DNA fragmentation and Fluoro-Jade B staining. The absence of activation of apoptotic cascade both in the P2 and cell accompanied with decreased DNA fragmentation and number of degenerating neurons clearly indicates that E treatment ensures the efficient protection against ischemic insult. Moreover, E-mediated modulation of pro-apoptotic signaling in the cortical cellular fractions involves cooperative activation of ERK and Akt, which may be implicated in the observed prevention of neurodegenerative changes.

17β-estradiol modulates mitochondrial Ca2+ flux in rat caudate nucleus and brain stem

The aim of this study was to examine the rapid non-genomic effect of 17β-estradiol (E2) on Ca(2+) transport in mitochondria isolated from the nerve terminals (synaptosomes) of caudate nuclei (NC) and brain stems (BS) of ovariectomised female rats. In physiological conditions no effect of E2 on Ca(2+) influx into synaptosomal mitochondria through ruthenium red (RR)-sensitive uniporter was observed. However, in the presence of uncoupling agent carbonyl cyanide4-(trifluoromethoxy)phenylhydrazone (FCCP) (1μmol/l), pre-treatment with 0.5nmol/l E2 protected mitochondrial membrane potential and consequently increased Ca(2+) influx (2.3-fold in NC and 3.1-fold in BS). At the same time, 0.5nmol/l E2 by increasing the affinity of mitochondrial Na(+)/Ca(2+) exchanger for Na(+) inhibited mitochondrial Ca(2+) efflux in NC and BS by about 40%. Also, the specific binding of physiological E2 concentrations (0.1-10nmol/l) to isolated synaptosomal mitochondria was detected. Using membrane impermeable E2 bound to bovine serum albumin and selective inhibitor of mitochondrial Na(+)/Ca(2+) exchanger, we obtained that E2s action on mitochondrial Ca(2+) efflux at least partially is due to the direct effects on the mitochondrial membrane and/or Na(+)/Ca(2+) exchanger located in inner mitochondrial membrane. Our results implicate E2 as a modulator of Ca(2+) concentration in mitochondrial matrix, and ultimately in the cytosol. Given the vital role of Ca(2+) in regulation of total nerve cells activity, especially energy metabolism, neurotransmission and directing the cells toward survival or cell death, the effects on mitochondrial Ca(2+) transport could be one of the important modes of E2 neuromodulatory action independent of the genome.

Inhibition of mitochondrial Na+-dependent Ca2+ efflux by 17β-estradiol in the rat hippocampus

Our results, as well as those of others, have indicated that 17β-estradiol (E2) exerts its nongenomic effects in neuronal cells by affecting plasma membrane Ca(2+) flux. In neuronal cells mitochondria possess Ca(2+) buffering properties as they both sequester and release Ca(2+). The goal of this study was to examine the rapid non-genomic effect of E2 on mitochondrial Ca(2+) transport in hippocampal synaptosomes from ovariectomised rats. In addition, we aimed to determine if, and to what extent, E2 receptors participated in mitochondrial Ca(2+) transport modulation by E2 in vitro. E2-specific binding and Ca(2+) transport was monitored. At physiological E2 concentrations (0.1-1.5 nmol/L), specific E2 binding to mitochondria isolated from hippocampal synaptosomes was detected with a B(max.) and K(m) of 37.6±2.6 fmol/mg protein and 0.69±0.14 nmol/L of free E2, respectively. The main mitochondrial Ca(2+) influx mechanism is the Ruthenium Red-sensitive uniporter driven by mitochondrial membrane potential. Despite no effect of E2 on Ca(2+) influx, a physiological E2 concentration (0.5 nmol/L) protected mitochondrial membrane potential and consequently Ca(2+) influx from the uncoupling agent carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (1 μmol/L). In neuronal cells the predominant mitochondrial Ca(2+) efflux mechanism is the Na(+)/Ca(2+) exchanger. E2 caused Ca(2+) efflux inhibition (by 46%) coupled with increased affinity of the Na(+)/Ca(2+) exchanger for Na(+). Using E2 receptor (ERα and ERβ) antagonists and agonists, we confirmed ERβs involvement in E2-induced mitochondrial membrane potential protection as well as Ca(2+) efflux inhibition. In summary, our results indicate that the non-genomic neuromodulatory role of E2 in rat hippocampus is achieved by affecting mitochondrial Ca(2+) transport via, in part, mitochondrial ERβ.

Inhibition of Vascular Endothelial Growth Factor Receptor 2 Exacerbates Loss of Lower Motor Neurons and Axons during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) are inflammatory demyelinating and neurodegenerative diseases in the central nervous system (CNS). It is believed that MS and EAE are initiated by autoreactive T lymphocytes that recognize myelin antigens; however, the mechanisms responsible for neurodegeneration in these diseases remain elusive. Data indicate that vascular endothelial growth factor A (VEGF-A) plays a role in the development of MS and EAE. Interestingly, VEGF-A is regarded as a neurotrophic factor in the CNS that promotes neuron survival and neurogenesis in various neurodegenerative diseases by activating VEGF receptor 2 (VEGFR2). In this study, we sought to explore the role of the VEGF-A/VEGFR2 signaling in neurodegeneration in MS and EAE. We showed that the expression of VEGF-A was decreased in the spinal cord during EAE and that VEGFR2 was activated in lower motor neurons in the spinal cord of EAE mice. Interestingly, we found that treatment with SU5416, a selective VEGFR2 inhibitor, starting after the onset of EAE clinical symptoms exacerbated lower motor neuron loss and axon loss in the lumbar spinal cord of mice undergoing EAE, but did not alter Purkinje neuron loss in the cerebellum or upper motor neuron loss in the cerebral cortex. Moreover, SU5416 treatment had a minimal effect on EAE clinical symptoms as well as inflammation, demyelination, and oligodendrocyte loss in the lumbar spinal cord. These results imply the protective effects of the VEGF-A/VEGFR2 signaling on lower motor neurons and axons in the spinal cord in MS and EAE.

Expression of ecto-nucleoside triphosphate diphosphohydrolase3 (NTPDase3) in the female rat brain during postnatal development.

Nucleoside triphosphate diphosphohydrolase3 (NTPDase3) is membrane-bound ecto-enzyme which hydrolyzes extracellular ATP, thus modulating the function of purinergic receptors and the pattern of purinergic signaling. Here we analyzed the developmental expression of NTPDase3 in female hypothalamus, cerebral cortex and hippocampal formation at different postnatal ages (PD7-PD90) by qRT-PCR and immunohistochemistry. In hypothalamus and hippocampus, a similar developmental profile was seen: NTPDase3 gene expression was stable during postnatal development and increased in adults. In the cortex, upregulation of NTPDase3 mRNA expression was seen at PD15 and further increase was evidenced in adults. Immunohistochemical analysis at PD7 revealed faint neuronal NTPDase3 localization in a dorsal hypothalamus. The immunoreactivity (ir) gradually increased in PD15 and PD20, in clusters of cells in the lateral, ventral and dorsomedial hypothalamus. Furthermore, in PD20 animals, NTPDase3-ir was detected on short fibers in the posterior hypothalamic area, while in PD30 the fibers appeared progressively longer and markedly varicose. In adults, the strongest NTPDase3-ir was observed in collections of cells in dorsomedial hypothalamic nucleus, dorsal and lateral hypothalamus and in several thalamic areas, whereas the varicose fibers traversed entire diencephalon, particularly paraventricular thalamic nucleus, ventromedial and dorsomedial hypothalamic nuclei, the arcuate nucleus and the prefornical part of the lateral hypothalamus. The presumably ascending NTPDase3-ir fibers were first observed in PD20; their density and the varicose appearance increased until the adulthood. Prominent enhancement of NTPDase3-ir in the hypothalamus coincides with age when animals acquire diurnal rhythms of sleeping and feeding, supporting the hypothesis that this enzyme may be involved in regulation of homeostatic functions.

Effects of chronic cerebral hypoperfusion and low-dose progesterone treatment on apoptotic processes, expression and subcellular localization of key elements within Akt and Erk signaling pathways in rat hippocampus.

The present study attempted to investigate how chronic cerebral hypoperfusion (CCH) and repeated low-dose progesterone (P) treatment affect gene and protein expression, subcellular distribution of key apoptotic elements within protein kinase B (Akt) and extracellular signal-regulated kinases (Erk) signal transduction pathways, as well as neurodegenerative processes and behavior. The results revealed the absence of Erk activation in CCH in cytosolic and synaptosomal fractions, indicating a lower threshold of Akt activation in brain ischemia, while P increased their levels above control values. CCH induced an increase in caspase 3 (Casp 3) and poly (ADP-ribose) polymerase (PARP) gene and protein expression. However, P restored expression of examined molecules in all observed fractions, except for the levels of Casp 3 in synapses which highlighted its possible non-apoptotic or even protective function. Our study showed the absence of nuclear factor kappa-light-chain-enhancer of activated b cells (NF-κB) response to this type of ischemic condition and its strong activation under the influence of P. Further, the initial increase in the number of apoptotic cells and amount of DNA fragmentation induced by CCH was significantly reduced by P. Finally, P reversed the CCH-induced reduction in locomotor activity, while promoting a substantial decrease in anxiety-related behavior. Our findings support the concept that repeated low-dose post-ischemic P treatment reduces CCH-induced neurodegeneration in the hippocampus. Neuroprotection is initiated through the activation of investigated kinases and regulation of their downstream molecules in subcellular specific manner, indicating that this treatment may be a promising therapy for alleviation of CCH-induced pathologies.

Time-related sex differences in cerebral hypoperfusion-induced brain injury

Although the model of cerebral hypoperfusion in rats has been a matter of many investigations over the years, the exact intracellular and biochemical mechanisms that lead to neuron loss and memory decline have not been clearly identified. In the current study, we examined whether cerebral hypoperfusion causes changes in hippocampal protein expression of apoptotic markers in the synaptosomal fraction and neurodegeneration in a time-dependent and sex-specific manner. Adult male and female Wistar rats were divided into two main groups, controls that underwent sham operation, and animals subjected to permanent bilateral occlusion of common carotid arteries. Both male and female rats were killed 3, 7 or 90 days following the insult. The obtained results indicate that the peak of processes that lead to apoptosis occured on postoperative day 7 and that they were more prominent in males, indicating that neuroprotective effects of certain substances (planned for future experiments), should be tested at this time point. [Projekat Ministarstva nauke Republike Srbije, br. 173044 i br. 41014]

Upregulation of Nucleoside Triphosphate Diphosphohydrolase-1 and Ecto-5′-Nucleotidase in Rat Hippocampus after Repeated Low-Dose Dexamethasone Administration

Although dexamethasone (DEX), a synthetic glucocorticoid receptor (GR) analog with profound effects on energy metabolism, immune system, and hypothalamic-pituitary-adrenal axis, is widely used therapeutically, its impact on the brain is poorly understood. The aim of the present study was to explore the effect of repeated low-dose DEX administration on the activity and expression of the ectonucleotidase enzymes which hydrolyze and therefore control extracellular ATP and adenosine concentrations in the synaptic cleft. Ectonucleotidases tested were ectonucleoside triphosphate diphosphohydrolase 1–3 (NTPDase1–3) and ecto-5′-nucleotidase (eN), whereas the effects were evaluated in two brain areas that show different sensitivity to glucocorticoid action, hippocampus, and cerebral cortex. In the hippocampus, but not in cerebral cortex, modest level of neurodegenerative changes as well as increase in ATP, ADP, and AMP hydrolysis and upregulation of NTPDase1 and eN mRNA expression ensued under the influence of DEX. The observed pattern of ectonucleotidase activation, which creates tissue volume with enhanced capacity for adenosine formation, is the hallmark of the response after different insults to the brain.

Cell proliferation assay – method optimisation for in vivo labeling of DNA in the rat forestomach

Abstract The study of cell proliferation is a useful tool in the fields of toxicology, pathophysiology and pharmacology. Cell proliferation and its degree can be evaluated using 5-bromo-2′-deoxyuridine which is incorporated into the newly synthesized DNA. The aim of this study was the optimization of subcutaneous application of 5-bromo-2′-deoxyuridine implantation for continuous and persistent marking of proliferating cells in the rat forestomach. 3-tert-Butyl-4-hydroxyanisole was used as the agent that ensures cell proliferation. In order to determine the optimal dose for proliferating cells labeling, 5-bromo-2′-deoxyuridine doses of 50 mg, 100 mg, 200 mg or 350 mg were implemented 2 days prior to sacrifice by flat-faced cylindrical matrices. Immunohistochemical analysis using 5-bromo-2′-deoxyuridine in situ detection kit was performed for the detection of 5-bromo-2′-deoxyuridine labeled cells. The results showed that for adult rats, the optimum 5-bromo-2′-deoxyuridine dose is 200 mg per animal for subcutaneous application. The here described manner of 5-bromo-2′-deoxyuridine in vivo labeling provides a simple, efficient, and reliable method for cell labeling, and at the same minimizes stress to animals.