Jaroslav Pavel - Academia.edu (original) (raw)
Papers by Jaroslav Pavel
Experimental and Therapeutic Medicine, 2018
The aim of the present study was to investigate the therapeutic efficacy of local hypothermia (be... more The aim of the present study was to investigate the therapeutic efficacy of local hypothermia (beginning 30 min post-injury persisting for 5 h) on tissue preservation along the rostro-caudal axis of the spinal cord (3 cm cranially and caudally from the lesion site), and the prevention of injury-induced functional loss in a newly developed computer-controlled compression model in minipig (force of impact 18N at L3 level), which mimics severe spinal cord injury (SCI). Minipigs underwent SCI with two post-injury modifications (durotomy vs. intact dura mater) followed by hypothermia through a perfusion chamber with cold (epidural t≈15˚C) saline, DMEM/F12 or enriched DMEM/F12 (SCI/durotomy group) and with room temperature (t≈24˚C) saline (SCI-only group). Minipigs treated with post-SCI durotomy demonstrated slower development of spontaneous neurological improvement at the early postinjury time points, although the outcome at 9 weeks of survival did not differ significantly between the two SCI groups. Hypothermia with saline (t≈15˚C) applied after SCI-durotomy improved white matter integrity in the dorsal and lateral columns in almost all rostro-caudal segments, whereas treatment with medium/enriched medium affected white matter integrity only in the rostral segments. Furthermore, regeneration of neurofilaments in the spinal cord after SCI-durotomy and hypothermic treatments indicated an important role of local saline hypothermia in the functional outcome. Although saline hypothermia (24˚C) in the SCI-only group exhibited a profound histological outcome (regarding the gray and white matter integrity and the number of motoneurons) and neurofilament protection in general, none of the tested treatments resulted in significant improvement of neurological status. The findings suggest that clinically-proven medical treatments for SCI combined with early 5 h-long saline hypothermia treatment without opening the dural sac could be more beneficial for tissue preservation and neurological outcome compared with hypothermia applied after durotomy.
International Journal of Molecular Sciences, 2018
The aim of our study was to limit the inflammatory response after a spinal cord injury (SCI) usin... more The aim of our study was to limit the inflammatory response after a spinal cord injury (SCI) using Atorvastatin (ATR), a potent inhibitor of cholesterol biosynthesis. Adult Wistar rats were divided into five experimental groups: one control group, two Th9 compression (40 g/15 min) groups, and two Th9 compression + ATR (5 mg/kg, i.p.) groups. The animals survived one day and six weeks. ATR applied in a single dose immediately post-SCI strongly reduced IL-1β release at 4 and 24 h and considerably reduced the activation of resident cells at one day post-injury. Acute ATR treatment effectively prevented the excessive infiltration of destructive M1 macrophages cranially, at the lesion site, and caudally (by 66%, 62%, and 52%, respectively) one day post-injury, whereas the infiltration of beneficial M2 macrophages was less affected (by 27%, 41%, and 16%). In addition, at the same time point, ATR visibly decreased caspase-3 cleavage in neurons, astrocytes, and oligodendrocytes. Six weeks post-SCI, ATR increased the expression of neurofilaments in the dorsolateral columns and Gap43-positive fibers in the lateral columns around the epicenter, and from day 30 to 42, significantly improved the motor activity of the hindlimbs. We suggest that early modulation of the inflammatory response via effects on the M1/M2 macrophages and the inhibition of caspase-3 expression could be crucial for the functional outcome.
Experimental and therapeutic medicine, 2018
This study investigated the neuroprotective efficacy of local hypothermia in a minipig model of s... more This study investigated the neuroprotective efficacy of local hypothermia in a minipig model of spinal cord injury (SCI) induced by a computer-controlled impactor device. The tissue integrity observed at the injury epicenter, and up to 3 cm cranially and caudally from the lesion site correlated with motor function. A computer-controlled device produced contusion lesions at L3 level with two different degrees of tissue sparing, depending upon pre-set impact parameters (8N- and 15N-force impact). Hypothermia with cold (4°C) saline or Dulbecco's modified Eagle's medium (DMEM)/F12 culture medium was applied 30 min after SCI (for 5 h) via a perfusion chamber (flow 2 ml/min). After saline hypothermia, the 8N-SCI group achieved faster recovery of hind limb function and the ability to walk from one to three steps at nine weeks in comparison with non-treated animals. Such improvements were not observed in saline-treated animals subjected to more severe 15N-SCI or in the group treated...
Regulatory peptides, Jan 27, 2009
To clarify the relationship between Angiotensin II AT(1) and AT(2) receptors, we studied AT(1) re... more To clarify the relationship between Angiotensin II AT(1) and AT(2) receptors, we studied AT(1) receptor mRNA and binding expression in tissues from AT(2) receptor gene disrupted (AT(2)(-/-)) female mice, where AT(2) receptors are not expressed in vivo, using in situ hybridization and quantitative autoradiography. Wild type mice expressed AT(1A) receptor mRNA and AT(1) receptor binding in lung parenchyma, the spleen, predominantly in the red pulp, and in liver parenchyma. In wild type mice, lung AT(2) receptors were expressed in lung bronchial epithelium and smooth muscle, and were not present in the lung parenchyma, the spleen or the liver. This indicates that AT(1) and AT(2) receptors were not expressed in the same cells. In AT(2)(-/-) mice, we found higher AT(1A) receptor mRNA and AT(1) receptor binding in lung parenchyma and in the red pulp of the spleen, but not in the liver, when compared to littermate wild type controls. Our results suggest that impaired AT(2) receptor functio...
Biologia. Section Cellular and Molecular Biology). In the present study we investigated the effec... more Biologia. Section Cellular and Molecular Biology). In the present study we investigated the effect of transient ischemia (13 and 17 min) followed by 1h and 3h of reperfusion on the Ca 2+-dependent nitric oxide synthase (NOS) activity. The enzyme activity was measured by the conversion of 14 [C]arginine to 14 [C]citrulline and determined in the dorsal horn, intermediate zone and ventral horn of the gray matter and in the dorsal, lateral and ventral columns of the white matter of the rabbit spinal cord. Transient ischemia induced by infrarenal balloon occlusion of the abdominal aorta for 13 min was not strong enough to cause significant changes in the catalytic NOS activity, although the experimental animals tended to show a mild deficit in hindlimb movement. The increase of enzyme activity was significant in the dorsal horn, dorsal and lateral column after 17 min ischemia. During subsequent reperfusions catalytic NOS activity undergoes a permanent increase in the dorsal horn and a transient increase in the intermediate zone. The neurological impairment of the hindlimbs, characterized by a partial or total paraplegia after 17 min ischemia and subsequent reperfusions may reflect differences in the integrity of spinal interneurons in the intermediate zone, i.e., the region being highly sensitive to transient spinal cord ischemia. Our results indicate that catalytic NOS activity may be regionally and temporally affected due to an ischemic insult and following subsequent reperfusions.
Stroke, 2006
Background and Purpose— Blockade of angiotensin II AT 1 receptors in cerebral microvessels protec... more Background and Purpose— Blockade of angiotensin II AT 1 receptors in cerebral microvessels protects against brain ischemia and inflammation. In this study, we tried to clarify the presence and regulation of the local renin-angiotensin system (RAS) in brain microvessels in hypertension. Methods— Spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) controls were treated with an AT 1 receptor antagonist (candesartan, 0.3 mg/kg per day) via subcutaneous osmotic minipumps for 4 weeks. The expression and localization of RAS components and the effect of AT 1 receptor blockade were assessed by Affymetrix microarray, qRT-PCR, Western blots, immunohistochemistry and immunofluorescence. Results— We found transcripts of most of RAS components in our microarray database, and confirmed their expression by qRT-PCR. Angiotensinogen (Aogen), angiotensin-converting enzyme (ACE) and AT 1 receptors were localized to the endothelium. There was no evidence of AT 2 receptor localization in the mic...
Stress, 2008
Spontaneously hypertensive rats, a stress-sensitive strain, were pretreated orally for 14 days wi... more Spontaneously hypertensive rats, a stress-sensitive strain, were pretreated orally for 14 days with the AT 1 receptor antagonist candesartan before submission to 2 h of cold-restraint stress. In non-treated rats, stress decreased AT 1 receptor binding in the median eminence and basolateral amygdala, increased AT 2 receptor binding in the medial subnucleus of the inferior olive, decreased AT 2 binding in the ventrolateral thalamic nucleus and increased tyrosine hydroxylase mRNA level in the locus coeruleus. In non-stressed rats, AT 1 receptor blockade reduced AT 1 receptor binding in all areas studied and enhanced AT 2 receptor binding in the medial subnucleus of the inferior olive. Candesartan pretreatment produced a similar decrease in brain AT 1 binding after stress, and prevented the stress-induced AT 2 receptor binding decrease in the ventrolateral thalamic nucleus. In the locus coeruleus and adrenal medulla, AT 1 blockade abolished the stress-induced increase in tyrosine hydroxylase mRNA level. Our results demonstrate that oral administration of candesartan effectively blocked brain AT 1 receptors, selectively increased central AT 2 receptor expression and prevented the stress-induced central stimulation of tyrosine hydroxylase transcription. The present results support a role of brain AT 1 and AT 2 receptors in the regulation of the stress response, and the hypothesis that AT 1 receptor antagonists may be considered as potential therapeutic compounds in stress related disorders in addition to their anti-hypertensive properties.
Regulatory Peptides, 2009
To clarify the role of Angiotensin II (Ang II) in the sensory system and especially in the trigem... more To clarify the role of Angiotensin II (Ang II) in the sensory system and especially in the trigeminal ganglia, we studied the expression of angiotensinogen (Ang-N)-, renin-, angiotensin converting enzyme (ACE)-and cathepsin D-mRNA, and the presence of Ang II and substance P in the rat and human trigeminal ganglia. The rat trigeminal ganglia expressed substantial amounts of Ang-N-and ACE mRNA as determined by quantitative real time PCR. Renin mRNA was untraceable in rat samples. Cathepsin D was detected in the rat trigeminal ganglia indicating the possibility of existence of pathways alternative to renin for Ang I formation. In situ hybridization in rat trigeminal ganglia revealed expression of Ang-N mRNA in the cytoplasm of numerous neurons. By using immunocytochemistry, a number of neurons and their processes in both the rat and human trigeminal ganglia were stained for Ang II. Post in situ hybridization immunocytochemistry reveals that in the rat trigeminal ganglia some, but not all Ang-N mRNA-positive neurons marked for Ang II. In some neurons Substance P was found colocalized with Ang II. Angiotensins from rat trigeminal ganglia were quantitated by radioimmunoassay with and without prior separation by high performance liquid chromatography. Immunoreactive angiotensin II (ir-Ang II) was consistently present and the sum of true Ang II (1-8) octapeptide and its specifically measured metabolites were found to account for it. Radioimmunological and immunocytochemical evidence of ir-Ang II in neuronal tissue is compatible with Ang II as a neurotransmitter. In conclusion, these results suggest that Ang II could be produced locally in the neurons of rat trigeminal ganglia. The localization and colocalization of neuronal Ang II with Substance P in the trigeminal ganglia neurons may be the basis for a participation and function of Ang II in the regulation of nociception and migraine pathology.
Neuropsychopharmacology, 2005
Long-term pretreatment with an angiotensin II AT 1 antagonist blocks angiotensin II effects in br... more Long-term pretreatment with an angiotensin II AT 1 antagonist blocks angiotensin II effects in brain and peripheral organs and abolishes the sympathoadrenal and hypothalamic-pituitary-adrenal responses to isolation stress. We determined whether AT 1 receptors were also important for the stress response of higher regulatory centers. We studied angiotensin II and corticotropin-releasing factor (CRF) receptors and benzodiazepine binding sites in brains of Wistar Hannover rats. Animals were pretreated for 13 days with vehicle or a central and peripheral AT 1 antagonist (candesartan, 0.5 mg/kg/day) via osmotic minipumps followed by 24 h of isolation in metabolic cages, or kept grouped throughout the study (grouped controls). In another study, we determined the influence of a similar treatment with candesartan on performance in an elevated plus-maze. AT 1 receptor blockade prevented the isolation-induced increase in brain AT 1 receptors and decrease in AT 2 binding in the locus coeruleus. AT 1 receptor antagonism also prevented the increase in tyrosine hydroxylase mRNA in the locus coeruleus. Pretreatment with the AT 1 receptor antagonist completely prevented the decrease in cortical CRF 1 receptor and benzodiazepine binding produced by isolation stress. In addition, pretreatment with candesartan increased the time spent in and the number of entries to open arms of the elevated plus-maze, measure of decreased anxiety. Our results implicate a modulation of upstream neurotransmission processes regulating cortical CRF 1 receptors and the GABA A complex as molecular mechanisms responsible for the anti-anxiety effect of centrally acting AT 1 receptor antagonists. We propose that AT 1 receptor antagonists can be considered as compounds with possible therapeutic anti-stress and anti-anxiety properties.
Neuropeptides, 2009
Angiotensin II (Ang II) and its type-1 receptor (AT 1) occur in neurons at multiple locations wit... more Angiotensin II (Ang II) and its type-1 receptor (AT 1) occur in neurons at multiple locations within the organism, but the basic biology of the receptor in the nervous system remains incompletely understood. We previously observed abundant AT 1-like binding sites and intense expression of AT 1 immunoreactivity in perikarya of the dorsal root ganglion and ventral horn of the rat spinal cord. We have now examined the receptor in rat sciatic nerve, including the dynamics of its axonal transport. Ligand-binding autoradiography of resting nerve showed "hot spots" of 125 I-Ang II binding that could be specifically blocked by the AT 1 antagonist, losartan. Immunohistochemistry with an AT 1-antibody validated by Western blots also showed patches of AT 1-reactivity in nerve. These patches were localized around large myelinated axons with faint immunoreactivity in their lumens. Sixteen hr after nerve ligation there was no change in the patches or hot spots, but luminal AT 1reactivity increased dramatically in a narrow zone immediately above the ligature. With double ligation there was a pronounced accumulation of AT 1 immunoreactivity proximal to the upstream ligature and a very slight accumulation distal to the second ligature. This asymmetric pattern of accumulation, confirmed by quantitative receptor binding autoradiography, probably reflected axonal transport rather than local production of receptor. Retrograde tracing and stereological analysis to determine the source of transported AT 1 indicated that many AT 1-positive fibers arise in the ventral horn, and a larger number arise in dorsal root ganglia. A corresponding result was obtained with double-label immunohistochemistry of ligated nerve, which showed AT 1 accumulations in both motor and sensory fibers. We conclude that somatic sensory and motor neurons of the rat export substantial quantities of AT 1 into axons, which transport them to the periphery. The physiologic implications of this finding require further investigation.
Neurological Research, 2008
The Journal of Physiological Sciences, 2009
This study was designed in order to consider whether the release of neuronally derived nitric oxi... more This study was designed in order to consider whether the release of neuronally derived nitric oxide (NO) in the lumbosacral spinal cord during ischemia/reperfusion is region-specific and whether changes in Ca 2?-dependent NO synthase (cNOS) activity paralell with functional outcome. The cNOS activity was measured in the spinal cord regions after 13-, 15-and 17-min ischemia alone and that followed by 24 h of reperfusion. In addition, the Tarlov's criteria were applied to define the neurological consequences of ischemia/ reperfusion in experimental animals. Based on the results, it is evident that only the 17-min ischemia alone was quite sufficient to cause changes in cNOS activity, however, without alterations in functional outcomes. On the other hand, the ischemic episodes followed by reperfusion caused dynamic, region-specific alterations in cNOS activity and consequently led to deterioration of motor function of hindlimbs in affected animals. Our results indicate that the motoneurons in the ventral horns respond more sensitively to ischemia/reperfusion than do neurons localized in the other spinal cord regions and that changes in cNOS activity may also influence the axonal conductance in the white matter and account for the impairment of motoneuronal activity in affected animals.
Journal of Neurochemistry, 2003
Few studies of lipid rafts have investigated gangliosides in brain tissue. This study focus on an... more Few studies of lipid rafts have investigated gangliosides in brain tissue. This study focus on analyses of lipids and the major brain gangliosides (GM1, GD1a, GD1b, GT1b) in human cortex (frontal, temporal) and corresponding detergent resistant membranes (DRMs), i.e. rafts. A high proportion of the gangliosides (18-26%) as well as of cholesterol (21%) and sphingomyelin (38%) was found in rafts, while lower yields was observed for ganglioside GM2 (9%), phospholipids (8%) and in particular proteins (2%). Significant alterations in lipid composition was noticed in rafts from Alzheimer brain tissue. These results show that sphingolipids and cholesterol are major constituents of rafts also in the human brain and that the main brain gangliosides are distributed in rafts to a similar degree. Moreover, lipid rafts might be considered in the pathology of Alzheimer's disease.
Journal of Hypertension, 2009
Objective-Inhibition of angiotensin II receptor type 1 (AT 1) reduces chronic inflammation associ... more Objective-Inhibition of angiotensin II receptor type 1 (AT 1) reduces chronic inflammation associated with hypertension. We asked whether AT 1 receptor inhibition would reduce the innate inflammatory response induced by bacterial lipopolysaccharide (LPS). Methods-We used unstimulated human circulating monocytes obtained from healthy donors by counterflow centrifugal elutriation. Monocytes were studied in vitro after incubation with LPS (50 ng/ml) with and without 1 μmol/l candesartan, an AT 1 receptor blocker. Angiotensin II receptor mRNA expression was determined by reverse transcriptase-PCR and receptor binding by autoradiography; inflammatory factor mRNA expression was studied by reverse transcriptase-PCR and cytokine release by ELISA. Results-Human monocytes did not express detectable AT 1 receptors, and angiotensin II did not induce inflammatory factor mRNA expression or cytokine release. However, candesartan substantially reduced the LPS-induced expression of the mRNAs for the LPS recognition protein cluster of differentiation 14, the proinflammatory cytokines tumor necrosis factor alpha, interleukin-1 beta and interleukin-6 and the lectin-like oxidized low-density lipoprotein receptor. In addition, candesartan reduced the activation of the nuclear factor kappa B pathway, the tumor necrosis factor alpha and interleukin-6 secretion, and the ROS formation induced by LPS, without affecting the secretion of interleukin-10. Conclusion-We hypothesize that the anti-inflammatory effects of candesartan in these cells are likely mediated by mechanisms unrelated to AT 1 receptor blockade. Our results demonstrate that candesartan significantly reduces the innate immune response to LPS in human circulating monocytes. The anti-inflammatory effects of candesartan may be of importance not only in hypertension but also in other inflammatory disorders.
Drug Development Research, 2005
The corticotropin-releasing factor (CRF), arginine-vasopressin (AVP), atrial natriuretic peptide ... more The corticotropin-releasing factor (CRF), arginine-vasopressin (AVP), atrial natriuretic peptide (ANP), and Angiotensin II (Ang II) interact, regulating the central sympathetic system, the hypothalamic-pituitary-adrenal (HPA) axis, and the central behavioral mechanisms involved in the response to stress and the development of mood disorders. Non-peptidic, orally active AVP V 1 , V 3 , or CRF 1 receptor antagonists reduce anxiety in animals. ANP is anxiolytic when administered to rodents and humans with anxiety disorders. These compounds are being developed for the therapy of anxiety and depression, but their clinical efficacy has not yet been established. Brain Ang II, a physiological ANP antagonist, promotes CRF and AVP release and stimulates the peripheral and central sympathetic systems. The degree of Ang II AT 1 receptor stimulation determines the response to stress. An orally active, non-peptidic Ang II AT 1 receptor antagonist blocks CRF and AVP release during stress, prevents the sympathoadrenal and hormonal stress reaction, the cortical CRF 1 and benzodiazepine receptor alterations, facilitates the effects of ANP, prevents stress-induced gastric ulcerations, and is anxiolytic in rodents. CRF, AVP, ANP, and the sympathetic system are intimately linked parts of a fundamental regulatory mechanism controlled by the brain Ang II system through AT 1 receptor stimulation, and should be studied together. Selective, safe antagonists of Ang II AT 1 receptors, with central effects after oral administration, have been actively used for the treatment of hypertension and should be considered also as potential anti-stress, anti-anxiety, and antidepressant compounds with properties similar or superior to the CRF and AVP antagonists or the ANP agonists under development.
Cellular and Molecular Neurobiology, 2006
1. Brief interruption of spinal cord blood flow resulting from transient abdominal aortic occlusi... more 1. Brief interruption of spinal cord blood flow resulting from transient abdominal aortic occlusion may lead to degeneration of specific spinal cord neurons and to irreversible loss of neurological function. The alteration of nitric oxide/nitric oxide synthase (NO/NOS) pool occurring after ischemic insult may play a protective or destructive role in neuronal survival of affected spinal cord segments. 2. In the present study, the spatiotemporal changes of NOS following transient ischemia were evaluated by investigating neuronal NOS immunoreactivity (nNOS-IR), reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry, and calcium-dependent NOS (cNOS) conversion of [(3)H] l-arginine to [(3)H] l-citrulline. 3. The greatest levels of these enzymes and activities were detected in the dorsal horn, which appeared to be most resistant to ischemia. In that area, the first significant increase in NADPHd staining and cNOS catalytic activity was found immediately after a 15-min ischemic insult. 4. Increases in the ventral horn were observed later (i.e., after a 24-h reperfusion period). While the most intense increase in nNOS-IR was detected in surviving motoneurons of animals with a shorter ischemic insult (13 min), the greatest increase of cNOS catalytic activity and NADPHd staining of the endothelial cells was found after stronger insult (15 min). 5. Given that the highest levels of nNOS, NADPHd, and cNOS were found in the ischemia-resistant dorsal horn, and nNOS-IR in surviving motoneurons, it is possible that NO production may play a neuroprotective role in ischemic/reperfusion injury.
Cellular and Molecular Neurobiology, 2006
1. The present study was designed to examine the nitric oxide synthase activities (constitutive a... more 1. The present study was designed to examine the nitric oxide synthase activities (constitutive and inducible) in the site of injury in response to Th10-Th11 spinal cord hemisection and, to determine whether unilateral disconnection of the spinal cord influences the NOS pools on the contra- and ipsilateral sides in segments located far away from the epicentre of injury. 2. A radioassay detection was used to determine Ca(2+)-dependent and inducible nitric oxide synthase activities. Somal, axonal and neuropil neuronal nitric oxide synthase was assessed by immunocytochemical study. A quantitative assessment of neuronal nitric oxide synthase immunoreactivity was made by an image analyser. The level of neuronal nitric oxide synthase protein was measured by the Western blot analysis. 3. Our data show the increase of inducible nitric oxide synthase activity and a decrease of Ca(2+)-dependent nitric oxide synthase activity in the injured site analysed 1 and 7 days after surgery. In segments remote from the epicentre of injury the inducible nitric oxide synthase activity was increased at both time points. Ca(2+)-dependent nitric oxide synthase activity had decreased in L5-S1 segments in a group of animals surviving for 7 days. A hemisection performed at thoracic level did not cause significant difference in the nitric oxide synthase activities and in the level of neuronal nitric oxide synthase protein between the contra- and ipsilateral sides in C6-Th1 and L5-S1 segments taken as a whole. Significant differences were observed, but only when the spinal cord was analysed segment by segment, and/or was divided into dorsal and ventral parts. The cell counts in the cervicothoracic (C7-Th1) and lumbosacral (L5-S1) enlargements revealed changes in neuronal nitric oxide synthase immunoreactivity on the ipsilateral side of the injury. The densitometric area measurements confirmed the reduction of somal, neuropil and axonal neuronal nitric oxide synthase immunoreactive staining in the ventral part of rostrally oriented segments. 4. Our findings provide evidence that the changes in nitric oxide synthase pools are limited not only to impact zone, but spread outside the original lesion. The regional distribution of nitric oxide synthase activity and neuronal nitric oxide synthase immunoreactivity, measured segment by segment shows that nitric oxide may play a significant role in the stepping cycle in the quadrupeds.
Cellular and Molecular Neurobiology, 2013
To clarify the role of angiotensin II (Ang II) in the regulation of sensory signaling, we studied... more To clarify the role of angiotensin II (Ang II) in the regulation of sensory signaling, we studied the effect of subpressor dose (150 ng/kg/min) of Ang II on pain-related behavior in relation with neuronal injury and activation of satellite glial cells (SGCs) in the dorsal root ganglia (DRGs) after chronic constriction injury (CCI). Systemic continuous delivery of Ang II induced the tactile, heat and cold hyperlagesia, when measured at 7 days ofpost-injury. Blockade of the AT 1 receptor with losartan (2.5 mg/kg/ day) prevented tactile hyperalgesia and attenuated cold hyperalgesia, but did not affect the response to noxious heat stimulus. A marked increase of large-sized injured primary afferent neurons, detected by ATF3 immunolabeling, was seen in lower lumbar DRGs on ipsilateral side after Ang II treatment. Subpressor dose of Ang II induced an increase of activated SGCs (detected by GFAP immunolabeling) enveloping large-diameter neurons. Our results suggested that Ang II through the AT 1 receptor activation is an important regulatory factor in neuropathic pain perception and plays an important role in the injury of largesized primary afferent neurons and activation of SGCs elicited by the CCI.
Cellular and Molecular Neurobiology, 2009
In addition to regulating blood pressure, Angiotensin II exerts powerful pro-inflammatory effects... more In addition to regulating blood pressure, Angiotensin II exerts powerful pro-inflammatory effects in hypertension through stimulation of its AT 1 receptors, most clearly demonstrated in peripheral arteries and in the cerebral vasculature. Administration of Angiotensin II receptor blockers (ARBs) decreases hypertension-related vascular inflammation in peripheral organs. In rodent models of genetic hypertension, ARBs reverse the inflammation in the cerebral microcirculation. We hypothesized that ARBs could be effective in inflammatory conditions beyond hypertension. Our more recent studies, summarized here, indicate that this is indeed the case. We used the model of systemic administration of the bacterial endotoxin lipopolysaccharide (LPS). LPS produces a robust initial inflammatory reaction, the innate immune response, in peripheral organs and in the brain. Pretreatment with the ARB candesartan significantly diminishes the response to LPS, including reduction of pro-inflammatory cytokine release to the general circulation and decreased production and release of the pro-inflammatory adrenal hormone aldosterone. In addition, the ARB very significantly decreased the LPS-induced gene expression of pro-inflammatory cytokines and microglia activation in the brain. Our results demonstrate that AT 1 receptor activity is essential for the unrestricted development of full-scale innate immune response in the periphery and in the brain. ARBs, due to their immune response-limiting properties, may be considered as therapeutically useful in a number of inflammatory diseases of the peripheral organs and the brain.
Cellular and Molecular Neurobiology, 2006
1. During the course of studies directed to determine the transport of Angiotensin II AT(2) recep... more 1. During the course of studies directed to determine the transport of Angiotensin II AT(2) receptors in the rat brain, we found that stab wounds to the brain revealed a binding site recognized by the AT(2) receptor ligand CGP42112 but not by Angiotensin II. 2. We localized this novel site to macrophages/microglia associated with physical or chemical injuries of the brain. 3. The non-Angiotensin II site was also highly localized to inflammatory lesions of peripheral arteries. 4. In rodent tissues, high binding expression was limited to the spleen and to circulating monocytes. A high-affinity binding site was also characterized in human monocytes. 5. Lack of affinity for many ligands binding to known macrophage receptors indicated the possibility that the non-Angiotensin II CGP42112 binding corresponds to a novel site.6. CGP42112 enhanced cell attachment to fibronectin and collagen and metalloproteinase-9 secretion from human monocytes incubated in serum-free medium but did not promote cytokine secretion. 7. When added in the presence of lipopolysaccharide, CGP42112 reduced the lipopolysaccharide-stimulated secretion of the pro-inflammatory cytokines TNF-alpha, IL-1, IL-1 beta, and IL-6, and increased protein kinase A. 8. Molecular modeling revealed that a CGP42112 derivative was selective for the novel macrophage site and did not recognize the Angiotensin II AT(2) receptor. 9. These results demonstrate that CGP42112, previously considered as a selective Angiotensin II AT(2) ligand, recognizes an additional non-Angiotensin II site different from AT(2) receptors. 10. Our observations indicate that CGP42112 or related molecules could be considered of interest as potential anti-inflammatory compounds.
Experimental and Therapeutic Medicine, 2018
The aim of the present study was to investigate the therapeutic efficacy of local hypothermia (be... more The aim of the present study was to investigate the therapeutic efficacy of local hypothermia (beginning 30 min post-injury persisting for 5 h) on tissue preservation along the rostro-caudal axis of the spinal cord (3 cm cranially and caudally from the lesion site), and the prevention of injury-induced functional loss in a newly developed computer-controlled compression model in minipig (force of impact 18N at L3 level), which mimics severe spinal cord injury (SCI). Minipigs underwent SCI with two post-injury modifications (durotomy vs. intact dura mater) followed by hypothermia through a perfusion chamber with cold (epidural t≈15˚C) saline, DMEM/F12 or enriched DMEM/F12 (SCI/durotomy group) and with room temperature (t≈24˚C) saline (SCI-only group). Minipigs treated with post-SCI durotomy demonstrated slower development of spontaneous neurological improvement at the early postinjury time points, although the outcome at 9 weeks of survival did not differ significantly between the two SCI groups. Hypothermia with saline (t≈15˚C) applied after SCI-durotomy improved white matter integrity in the dorsal and lateral columns in almost all rostro-caudal segments, whereas treatment with medium/enriched medium affected white matter integrity only in the rostral segments. Furthermore, regeneration of neurofilaments in the spinal cord after SCI-durotomy and hypothermic treatments indicated an important role of local saline hypothermia in the functional outcome. Although saline hypothermia (24˚C) in the SCI-only group exhibited a profound histological outcome (regarding the gray and white matter integrity and the number of motoneurons) and neurofilament protection in general, none of the tested treatments resulted in significant improvement of neurological status. The findings suggest that clinically-proven medical treatments for SCI combined with early 5 h-long saline hypothermia treatment without opening the dural sac could be more beneficial for tissue preservation and neurological outcome compared with hypothermia applied after durotomy.
International Journal of Molecular Sciences, 2018
The aim of our study was to limit the inflammatory response after a spinal cord injury (SCI) usin... more The aim of our study was to limit the inflammatory response after a spinal cord injury (SCI) using Atorvastatin (ATR), a potent inhibitor of cholesterol biosynthesis. Adult Wistar rats were divided into five experimental groups: one control group, two Th9 compression (40 g/15 min) groups, and two Th9 compression + ATR (5 mg/kg, i.p.) groups. The animals survived one day and six weeks. ATR applied in a single dose immediately post-SCI strongly reduced IL-1β release at 4 and 24 h and considerably reduced the activation of resident cells at one day post-injury. Acute ATR treatment effectively prevented the excessive infiltration of destructive M1 macrophages cranially, at the lesion site, and caudally (by 66%, 62%, and 52%, respectively) one day post-injury, whereas the infiltration of beneficial M2 macrophages was less affected (by 27%, 41%, and 16%). In addition, at the same time point, ATR visibly decreased caspase-3 cleavage in neurons, astrocytes, and oligodendrocytes. Six weeks post-SCI, ATR increased the expression of neurofilaments in the dorsolateral columns and Gap43-positive fibers in the lateral columns around the epicenter, and from day 30 to 42, significantly improved the motor activity of the hindlimbs. We suggest that early modulation of the inflammatory response via effects on the M1/M2 macrophages and the inhibition of caspase-3 expression could be crucial for the functional outcome.
Experimental and therapeutic medicine, 2018
This study investigated the neuroprotective efficacy of local hypothermia in a minipig model of s... more This study investigated the neuroprotective efficacy of local hypothermia in a minipig model of spinal cord injury (SCI) induced by a computer-controlled impactor device. The tissue integrity observed at the injury epicenter, and up to 3 cm cranially and caudally from the lesion site correlated with motor function. A computer-controlled device produced contusion lesions at L3 level with two different degrees of tissue sparing, depending upon pre-set impact parameters (8N- and 15N-force impact). Hypothermia with cold (4°C) saline or Dulbecco's modified Eagle's medium (DMEM)/F12 culture medium was applied 30 min after SCI (for 5 h) via a perfusion chamber (flow 2 ml/min). After saline hypothermia, the 8N-SCI group achieved faster recovery of hind limb function and the ability to walk from one to three steps at nine weeks in comparison with non-treated animals. Such improvements were not observed in saline-treated animals subjected to more severe 15N-SCI or in the group treated...
Regulatory peptides, Jan 27, 2009
To clarify the relationship between Angiotensin II AT(1) and AT(2) receptors, we studied AT(1) re... more To clarify the relationship between Angiotensin II AT(1) and AT(2) receptors, we studied AT(1) receptor mRNA and binding expression in tissues from AT(2) receptor gene disrupted (AT(2)(-/-)) female mice, where AT(2) receptors are not expressed in vivo, using in situ hybridization and quantitative autoradiography. Wild type mice expressed AT(1A) receptor mRNA and AT(1) receptor binding in lung parenchyma, the spleen, predominantly in the red pulp, and in liver parenchyma. In wild type mice, lung AT(2) receptors were expressed in lung bronchial epithelium and smooth muscle, and were not present in the lung parenchyma, the spleen or the liver. This indicates that AT(1) and AT(2) receptors were not expressed in the same cells. In AT(2)(-/-) mice, we found higher AT(1A) receptor mRNA and AT(1) receptor binding in lung parenchyma and in the red pulp of the spleen, but not in the liver, when compared to littermate wild type controls. Our results suggest that impaired AT(2) receptor functio...
Biologia. Section Cellular and Molecular Biology). In the present study we investigated the effec... more Biologia. Section Cellular and Molecular Biology). In the present study we investigated the effect of transient ischemia (13 and 17 min) followed by 1h and 3h of reperfusion on the Ca 2+-dependent nitric oxide synthase (NOS) activity. The enzyme activity was measured by the conversion of 14 [C]arginine to 14 [C]citrulline and determined in the dorsal horn, intermediate zone and ventral horn of the gray matter and in the dorsal, lateral and ventral columns of the white matter of the rabbit spinal cord. Transient ischemia induced by infrarenal balloon occlusion of the abdominal aorta for 13 min was not strong enough to cause significant changes in the catalytic NOS activity, although the experimental animals tended to show a mild deficit in hindlimb movement. The increase of enzyme activity was significant in the dorsal horn, dorsal and lateral column after 17 min ischemia. During subsequent reperfusions catalytic NOS activity undergoes a permanent increase in the dorsal horn and a transient increase in the intermediate zone. The neurological impairment of the hindlimbs, characterized by a partial or total paraplegia after 17 min ischemia and subsequent reperfusions may reflect differences in the integrity of spinal interneurons in the intermediate zone, i.e., the region being highly sensitive to transient spinal cord ischemia. Our results indicate that catalytic NOS activity may be regionally and temporally affected due to an ischemic insult and following subsequent reperfusions.
Stroke, 2006
Background and Purpose— Blockade of angiotensin II AT 1 receptors in cerebral microvessels protec... more Background and Purpose— Blockade of angiotensin II AT 1 receptors in cerebral microvessels protects against brain ischemia and inflammation. In this study, we tried to clarify the presence and regulation of the local renin-angiotensin system (RAS) in brain microvessels in hypertension. Methods— Spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) controls were treated with an AT 1 receptor antagonist (candesartan, 0.3 mg/kg per day) via subcutaneous osmotic minipumps for 4 weeks. The expression and localization of RAS components and the effect of AT 1 receptor blockade were assessed by Affymetrix microarray, qRT-PCR, Western blots, immunohistochemistry and immunofluorescence. Results— We found transcripts of most of RAS components in our microarray database, and confirmed their expression by qRT-PCR. Angiotensinogen (Aogen), angiotensin-converting enzyme (ACE) and AT 1 receptors were localized to the endothelium. There was no evidence of AT 2 receptor localization in the mic...
Stress, 2008
Spontaneously hypertensive rats, a stress-sensitive strain, were pretreated orally for 14 days wi... more Spontaneously hypertensive rats, a stress-sensitive strain, were pretreated orally for 14 days with the AT 1 receptor antagonist candesartan before submission to 2 h of cold-restraint stress. In non-treated rats, stress decreased AT 1 receptor binding in the median eminence and basolateral amygdala, increased AT 2 receptor binding in the medial subnucleus of the inferior olive, decreased AT 2 binding in the ventrolateral thalamic nucleus and increased tyrosine hydroxylase mRNA level in the locus coeruleus. In non-stressed rats, AT 1 receptor blockade reduced AT 1 receptor binding in all areas studied and enhanced AT 2 receptor binding in the medial subnucleus of the inferior olive. Candesartan pretreatment produced a similar decrease in brain AT 1 binding after stress, and prevented the stress-induced AT 2 receptor binding decrease in the ventrolateral thalamic nucleus. In the locus coeruleus and adrenal medulla, AT 1 blockade abolished the stress-induced increase in tyrosine hydroxylase mRNA level. Our results demonstrate that oral administration of candesartan effectively blocked brain AT 1 receptors, selectively increased central AT 2 receptor expression and prevented the stress-induced central stimulation of tyrosine hydroxylase transcription. The present results support a role of brain AT 1 and AT 2 receptors in the regulation of the stress response, and the hypothesis that AT 1 receptor antagonists may be considered as potential therapeutic compounds in stress related disorders in addition to their anti-hypertensive properties.
Regulatory Peptides, 2009
To clarify the role of Angiotensin II (Ang II) in the sensory system and especially in the trigem... more To clarify the role of Angiotensin II (Ang II) in the sensory system and especially in the trigeminal ganglia, we studied the expression of angiotensinogen (Ang-N)-, renin-, angiotensin converting enzyme (ACE)-and cathepsin D-mRNA, and the presence of Ang II and substance P in the rat and human trigeminal ganglia. The rat trigeminal ganglia expressed substantial amounts of Ang-N-and ACE mRNA as determined by quantitative real time PCR. Renin mRNA was untraceable in rat samples. Cathepsin D was detected in the rat trigeminal ganglia indicating the possibility of existence of pathways alternative to renin for Ang I formation. In situ hybridization in rat trigeminal ganglia revealed expression of Ang-N mRNA in the cytoplasm of numerous neurons. By using immunocytochemistry, a number of neurons and their processes in both the rat and human trigeminal ganglia were stained for Ang II. Post in situ hybridization immunocytochemistry reveals that in the rat trigeminal ganglia some, but not all Ang-N mRNA-positive neurons marked for Ang II. In some neurons Substance P was found colocalized with Ang II. Angiotensins from rat trigeminal ganglia were quantitated by radioimmunoassay with and without prior separation by high performance liquid chromatography. Immunoreactive angiotensin II (ir-Ang II) was consistently present and the sum of true Ang II (1-8) octapeptide and its specifically measured metabolites were found to account for it. Radioimmunological and immunocytochemical evidence of ir-Ang II in neuronal tissue is compatible with Ang II as a neurotransmitter. In conclusion, these results suggest that Ang II could be produced locally in the neurons of rat trigeminal ganglia. The localization and colocalization of neuronal Ang II with Substance P in the trigeminal ganglia neurons may be the basis for a participation and function of Ang II in the regulation of nociception and migraine pathology.
Neuropsychopharmacology, 2005
Long-term pretreatment with an angiotensin II AT 1 antagonist blocks angiotensin II effects in br... more Long-term pretreatment with an angiotensin II AT 1 antagonist blocks angiotensin II effects in brain and peripheral organs and abolishes the sympathoadrenal and hypothalamic-pituitary-adrenal responses to isolation stress. We determined whether AT 1 receptors were also important for the stress response of higher regulatory centers. We studied angiotensin II and corticotropin-releasing factor (CRF) receptors and benzodiazepine binding sites in brains of Wistar Hannover rats. Animals were pretreated for 13 days with vehicle or a central and peripheral AT 1 antagonist (candesartan, 0.5 mg/kg/day) via osmotic minipumps followed by 24 h of isolation in metabolic cages, or kept grouped throughout the study (grouped controls). In another study, we determined the influence of a similar treatment with candesartan on performance in an elevated plus-maze. AT 1 receptor blockade prevented the isolation-induced increase in brain AT 1 receptors and decrease in AT 2 binding in the locus coeruleus. AT 1 receptor antagonism also prevented the increase in tyrosine hydroxylase mRNA in the locus coeruleus. Pretreatment with the AT 1 receptor antagonist completely prevented the decrease in cortical CRF 1 receptor and benzodiazepine binding produced by isolation stress. In addition, pretreatment with candesartan increased the time spent in and the number of entries to open arms of the elevated plus-maze, measure of decreased anxiety. Our results implicate a modulation of upstream neurotransmission processes regulating cortical CRF 1 receptors and the GABA A complex as molecular mechanisms responsible for the anti-anxiety effect of centrally acting AT 1 receptor antagonists. We propose that AT 1 receptor antagonists can be considered as compounds with possible therapeutic anti-stress and anti-anxiety properties.
Neuropeptides, 2009
Angiotensin II (Ang II) and its type-1 receptor (AT 1) occur in neurons at multiple locations wit... more Angiotensin II (Ang II) and its type-1 receptor (AT 1) occur in neurons at multiple locations within the organism, but the basic biology of the receptor in the nervous system remains incompletely understood. We previously observed abundant AT 1-like binding sites and intense expression of AT 1 immunoreactivity in perikarya of the dorsal root ganglion and ventral horn of the rat spinal cord. We have now examined the receptor in rat sciatic nerve, including the dynamics of its axonal transport. Ligand-binding autoradiography of resting nerve showed "hot spots" of 125 I-Ang II binding that could be specifically blocked by the AT 1 antagonist, losartan. Immunohistochemistry with an AT 1-antibody validated by Western blots also showed patches of AT 1-reactivity in nerve. These patches were localized around large myelinated axons with faint immunoreactivity in their lumens. Sixteen hr after nerve ligation there was no change in the patches or hot spots, but luminal AT 1reactivity increased dramatically in a narrow zone immediately above the ligature. With double ligation there was a pronounced accumulation of AT 1 immunoreactivity proximal to the upstream ligature and a very slight accumulation distal to the second ligature. This asymmetric pattern of accumulation, confirmed by quantitative receptor binding autoradiography, probably reflected axonal transport rather than local production of receptor. Retrograde tracing and stereological analysis to determine the source of transported AT 1 indicated that many AT 1-positive fibers arise in the ventral horn, and a larger number arise in dorsal root ganglia. A corresponding result was obtained with double-label immunohistochemistry of ligated nerve, which showed AT 1 accumulations in both motor and sensory fibers. We conclude that somatic sensory and motor neurons of the rat export substantial quantities of AT 1 into axons, which transport them to the periphery. The physiologic implications of this finding require further investigation.
Neurological Research, 2008
The Journal of Physiological Sciences, 2009
This study was designed in order to consider whether the release of neuronally derived nitric oxi... more This study was designed in order to consider whether the release of neuronally derived nitric oxide (NO) in the lumbosacral spinal cord during ischemia/reperfusion is region-specific and whether changes in Ca 2?-dependent NO synthase (cNOS) activity paralell with functional outcome. The cNOS activity was measured in the spinal cord regions after 13-, 15-and 17-min ischemia alone and that followed by 24 h of reperfusion. In addition, the Tarlov's criteria were applied to define the neurological consequences of ischemia/ reperfusion in experimental animals. Based on the results, it is evident that only the 17-min ischemia alone was quite sufficient to cause changes in cNOS activity, however, without alterations in functional outcomes. On the other hand, the ischemic episodes followed by reperfusion caused dynamic, region-specific alterations in cNOS activity and consequently led to deterioration of motor function of hindlimbs in affected animals. Our results indicate that the motoneurons in the ventral horns respond more sensitively to ischemia/reperfusion than do neurons localized in the other spinal cord regions and that changes in cNOS activity may also influence the axonal conductance in the white matter and account for the impairment of motoneuronal activity in affected animals.
Journal of Neurochemistry, 2003
Few studies of lipid rafts have investigated gangliosides in brain tissue. This study focus on an... more Few studies of lipid rafts have investigated gangliosides in brain tissue. This study focus on analyses of lipids and the major brain gangliosides (GM1, GD1a, GD1b, GT1b) in human cortex (frontal, temporal) and corresponding detergent resistant membranes (DRMs), i.e. rafts. A high proportion of the gangliosides (18-26%) as well as of cholesterol (21%) and sphingomyelin (38%) was found in rafts, while lower yields was observed for ganglioside GM2 (9%), phospholipids (8%) and in particular proteins (2%). Significant alterations in lipid composition was noticed in rafts from Alzheimer brain tissue. These results show that sphingolipids and cholesterol are major constituents of rafts also in the human brain and that the main brain gangliosides are distributed in rafts to a similar degree. Moreover, lipid rafts might be considered in the pathology of Alzheimer's disease.
Journal of Hypertension, 2009
Objective-Inhibition of angiotensin II receptor type 1 (AT 1) reduces chronic inflammation associ... more Objective-Inhibition of angiotensin II receptor type 1 (AT 1) reduces chronic inflammation associated with hypertension. We asked whether AT 1 receptor inhibition would reduce the innate inflammatory response induced by bacterial lipopolysaccharide (LPS). Methods-We used unstimulated human circulating monocytes obtained from healthy donors by counterflow centrifugal elutriation. Monocytes were studied in vitro after incubation with LPS (50 ng/ml) with and without 1 μmol/l candesartan, an AT 1 receptor blocker. Angiotensin II receptor mRNA expression was determined by reverse transcriptase-PCR and receptor binding by autoradiography; inflammatory factor mRNA expression was studied by reverse transcriptase-PCR and cytokine release by ELISA. Results-Human monocytes did not express detectable AT 1 receptors, and angiotensin II did not induce inflammatory factor mRNA expression or cytokine release. However, candesartan substantially reduced the LPS-induced expression of the mRNAs for the LPS recognition protein cluster of differentiation 14, the proinflammatory cytokines tumor necrosis factor alpha, interleukin-1 beta and interleukin-6 and the lectin-like oxidized low-density lipoprotein receptor. In addition, candesartan reduced the activation of the nuclear factor kappa B pathway, the tumor necrosis factor alpha and interleukin-6 secretion, and the ROS formation induced by LPS, without affecting the secretion of interleukin-10. Conclusion-We hypothesize that the anti-inflammatory effects of candesartan in these cells are likely mediated by mechanisms unrelated to AT 1 receptor blockade. Our results demonstrate that candesartan significantly reduces the innate immune response to LPS in human circulating monocytes. The anti-inflammatory effects of candesartan may be of importance not only in hypertension but also in other inflammatory disorders.
Drug Development Research, 2005
The corticotropin-releasing factor (CRF), arginine-vasopressin (AVP), atrial natriuretic peptide ... more The corticotropin-releasing factor (CRF), arginine-vasopressin (AVP), atrial natriuretic peptide (ANP), and Angiotensin II (Ang II) interact, regulating the central sympathetic system, the hypothalamic-pituitary-adrenal (HPA) axis, and the central behavioral mechanisms involved in the response to stress and the development of mood disorders. Non-peptidic, orally active AVP V 1 , V 3 , or CRF 1 receptor antagonists reduce anxiety in animals. ANP is anxiolytic when administered to rodents and humans with anxiety disorders. These compounds are being developed for the therapy of anxiety and depression, but their clinical efficacy has not yet been established. Brain Ang II, a physiological ANP antagonist, promotes CRF and AVP release and stimulates the peripheral and central sympathetic systems. The degree of Ang II AT 1 receptor stimulation determines the response to stress. An orally active, non-peptidic Ang II AT 1 receptor antagonist blocks CRF and AVP release during stress, prevents the sympathoadrenal and hormonal stress reaction, the cortical CRF 1 and benzodiazepine receptor alterations, facilitates the effects of ANP, prevents stress-induced gastric ulcerations, and is anxiolytic in rodents. CRF, AVP, ANP, and the sympathetic system are intimately linked parts of a fundamental regulatory mechanism controlled by the brain Ang II system through AT 1 receptor stimulation, and should be studied together. Selective, safe antagonists of Ang II AT 1 receptors, with central effects after oral administration, have been actively used for the treatment of hypertension and should be considered also as potential anti-stress, anti-anxiety, and antidepressant compounds with properties similar or superior to the CRF and AVP antagonists or the ANP agonists under development.
Cellular and Molecular Neurobiology, 2006
1. Brief interruption of spinal cord blood flow resulting from transient abdominal aortic occlusi... more 1. Brief interruption of spinal cord blood flow resulting from transient abdominal aortic occlusion may lead to degeneration of specific spinal cord neurons and to irreversible loss of neurological function. The alteration of nitric oxide/nitric oxide synthase (NO/NOS) pool occurring after ischemic insult may play a protective or destructive role in neuronal survival of affected spinal cord segments. 2. In the present study, the spatiotemporal changes of NOS following transient ischemia were evaluated by investigating neuronal NOS immunoreactivity (nNOS-IR), reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry, and calcium-dependent NOS (cNOS) conversion of [(3)H] l-arginine to [(3)H] l-citrulline. 3. The greatest levels of these enzymes and activities were detected in the dorsal horn, which appeared to be most resistant to ischemia. In that area, the first significant increase in NADPHd staining and cNOS catalytic activity was found immediately after a 15-min ischemic insult. 4. Increases in the ventral horn were observed later (i.e., after a 24-h reperfusion period). While the most intense increase in nNOS-IR was detected in surviving motoneurons of animals with a shorter ischemic insult (13 min), the greatest increase of cNOS catalytic activity and NADPHd staining of the endothelial cells was found after stronger insult (15 min). 5. Given that the highest levels of nNOS, NADPHd, and cNOS were found in the ischemia-resistant dorsal horn, and nNOS-IR in surviving motoneurons, it is possible that NO production may play a neuroprotective role in ischemic/reperfusion injury.
Cellular and Molecular Neurobiology, 2006
1. The present study was designed to examine the nitric oxide synthase activities (constitutive a... more 1. The present study was designed to examine the nitric oxide synthase activities (constitutive and inducible) in the site of injury in response to Th10-Th11 spinal cord hemisection and, to determine whether unilateral disconnection of the spinal cord influences the NOS pools on the contra- and ipsilateral sides in segments located far away from the epicentre of injury. 2. A radioassay detection was used to determine Ca(2+)-dependent and inducible nitric oxide synthase activities. Somal, axonal and neuropil neuronal nitric oxide synthase was assessed by immunocytochemical study. A quantitative assessment of neuronal nitric oxide synthase immunoreactivity was made by an image analyser. The level of neuronal nitric oxide synthase protein was measured by the Western blot analysis. 3. Our data show the increase of inducible nitric oxide synthase activity and a decrease of Ca(2+)-dependent nitric oxide synthase activity in the injured site analysed 1 and 7 days after surgery. In segments remote from the epicentre of injury the inducible nitric oxide synthase activity was increased at both time points. Ca(2+)-dependent nitric oxide synthase activity had decreased in L5-S1 segments in a group of animals surviving for 7 days. A hemisection performed at thoracic level did not cause significant difference in the nitric oxide synthase activities and in the level of neuronal nitric oxide synthase protein between the contra- and ipsilateral sides in C6-Th1 and L5-S1 segments taken as a whole. Significant differences were observed, but only when the spinal cord was analysed segment by segment, and/or was divided into dorsal and ventral parts. The cell counts in the cervicothoracic (C7-Th1) and lumbosacral (L5-S1) enlargements revealed changes in neuronal nitric oxide synthase immunoreactivity on the ipsilateral side of the injury. The densitometric area measurements confirmed the reduction of somal, neuropil and axonal neuronal nitric oxide synthase immunoreactive staining in the ventral part of rostrally oriented segments. 4. Our findings provide evidence that the changes in nitric oxide synthase pools are limited not only to impact zone, but spread outside the original lesion. The regional distribution of nitric oxide synthase activity and neuronal nitric oxide synthase immunoreactivity, measured segment by segment shows that nitric oxide may play a significant role in the stepping cycle in the quadrupeds.
Cellular and Molecular Neurobiology, 2013
To clarify the role of angiotensin II (Ang II) in the regulation of sensory signaling, we studied... more To clarify the role of angiotensin II (Ang II) in the regulation of sensory signaling, we studied the effect of subpressor dose (150 ng/kg/min) of Ang II on pain-related behavior in relation with neuronal injury and activation of satellite glial cells (SGCs) in the dorsal root ganglia (DRGs) after chronic constriction injury (CCI). Systemic continuous delivery of Ang II induced the tactile, heat and cold hyperlagesia, when measured at 7 days ofpost-injury. Blockade of the AT 1 receptor with losartan (2.5 mg/kg/ day) prevented tactile hyperalgesia and attenuated cold hyperalgesia, but did not affect the response to noxious heat stimulus. A marked increase of large-sized injured primary afferent neurons, detected by ATF3 immunolabeling, was seen in lower lumbar DRGs on ipsilateral side after Ang II treatment. Subpressor dose of Ang II induced an increase of activated SGCs (detected by GFAP immunolabeling) enveloping large-diameter neurons. Our results suggested that Ang II through the AT 1 receptor activation is an important regulatory factor in neuropathic pain perception and plays an important role in the injury of largesized primary afferent neurons and activation of SGCs elicited by the CCI.
Cellular and Molecular Neurobiology, 2009
In addition to regulating blood pressure, Angiotensin II exerts powerful pro-inflammatory effects... more In addition to regulating blood pressure, Angiotensin II exerts powerful pro-inflammatory effects in hypertension through stimulation of its AT 1 receptors, most clearly demonstrated in peripheral arteries and in the cerebral vasculature. Administration of Angiotensin II receptor blockers (ARBs) decreases hypertension-related vascular inflammation in peripheral organs. In rodent models of genetic hypertension, ARBs reverse the inflammation in the cerebral microcirculation. We hypothesized that ARBs could be effective in inflammatory conditions beyond hypertension. Our more recent studies, summarized here, indicate that this is indeed the case. We used the model of systemic administration of the bacterial endotoxin lipopolysaccharide (LPS). LPS produces a robust initial inflammatory reaction, the innate immune response, in peripheral organs and in the brain. Pretreatment with the ARB candesartan significantly diminishes the response to LPS, including reduction of pro-inflammatory cytokine release to the general circulation and decreased production and release of the pro-inflammatory adrenal hormone aldosterone. In addition, the ARB very significantly decreased the LPS-induced gene expression of pro-inflammatory cytokines and microglia activation in the brain. Our results demonstrate that AT 1 receptor activity is essential for the unrestricted development of full-scale innate immune response in the periphery and in the brain. ARBs, due to their immune response-limiting properties, may be considered as therapeutically useful in a number of inflammatory diseases of the peripheral organs and the brain.
Cellular and Molecular Neurobiology, 2006
1. During the course of studies directed to determine the transport of Angiotensin II AT(2) recep... more 1. During the course of studies directed to determine the transport of Angiotensin II AT(2) receptors in the rat brain, we found that stab wounds to the brain revealed a binding site recognized by the AT(2) receptor ligand CGP42112 but not by Angiotensin II. 2. We localized this novel site to macrophages/microglia associated with physical or chemical injuries of the brain. 3. The non-Angiotensin II site was also highly localized to inflammatory lesions of peripheral arteries. 4. In rodent tissues, high binding expression was limited to the spleen and to circulating monocytes. A high-affinity binding site was also characterized in human monocytes. 5. Lack of affinity for many ligands binding to known macrophage receptors indicated the possibility that the non-Angiotensin II CGP42112 binding corresponds to a novel site.6. CGP42112 enhanced cell attachment to fibronectin and collagen and metalloproteinase-9 secretion from human monocytes incubated in serum-free medium but did not promote cytokine secretion. 7. When added in the presence of lipopolysaccharide, CGP42112 reduced the lipopolysaccharide-stimulated secretion of the pro-inflammatory cytokines TNF-alpha, IL-1, IL-1 beta, and IL-6, and increased protein kinase A. 8. Molecular modeling revealed that a CGP42112 derivative was selective for the novel macrophage site and did not recognize the Angiotensin II AT(2) receptor. 9. These results demonstrate that CGP42112, previously considered as a selective Angiotensin II AT(2) ligand, recognizes an additional non-Angiotensin II site different from AT(2) receptors. 10. Our observations indicate that CGP42112 or related molecules could be considered of interest as potential anti-inflammatory compounds.