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Spontaneous recovery of lost motor functions is relative fast in rodent models after inducing a very mild/moderate spinal cord injury (SCI), and this may complicate a reliable evaluation of the effectiveness of potential therapy. Therefore, a severe graded (30 g, 40 g and 50 g) weight-compression SCI at the Th9 spinal segment, involving an acute mechanical impact followed by 15 min of persistent compression, was studied in adult female Wistar rats. Functional parameters, such as spontaneous recovery of motor hind limb and bladder emptying function, and the presence of hematuria were evaluated within 28 days of the post-traumatic period. The disruption of the blood-spinal cord barrier, measured by extravasated Evans Blue dye, was examined 24 h after the SCI, when maximum permeability occurs. At the end of the survival period, the degradation of gray and white matter associated with the formation of cystic cavities, and quantitative changes of glial structural proteins, such as GFAP, and integral components of axonal architecture, such as neurofilaments and myelin basic protein, were evaluated in the lesioned area of the spinal cord. Based on these functional and histological parameters, and taking the animal’s welfare into account, the 40 g weight can be considered as an upper limit for severe traumatic injury in this compression model.

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.

Spontaneous recovery of lost motor functions is relative fast in rodent models after inducing a very mild/moderate spinal cord injury (SCI), and this may complicate a reliable evaluation of the effectiveness of potential therapy. Therefore, a severe graded (30 g, 40 g and 50 g) weight-compression SCI at the Th9 spinal segment, involving an acute mechanical impact followed by 15 min of persistent compression, was studied in adult female Wistar rats. Functional parameters, such as spontaneous recovery of motor hind limb and bladder emptying function, and the presence of hematuria were evaluated within 28 days of the post-traumatic period. The disruption of the blood-spinal cord barrier, measured by extravasated Evans Blue dye, was examined 24 h after the SCI, when maximum permeability occurs. At the end of the survival period, the degradation of gray and white matter associated with the formation of cystic cavities, and quantitative changes of glial structural proteins, such as GFAP, and integral components of axonal architecture, such as neurofilaments and myelin basic protein, were evaluated in the lesioned area of the spinal cord. Based on these functional and histological parameters, and taking the animal's welfare into account, the 40 g weight can be considered as an upper limit for severe traumatic injury in this compression model.

To clarify the role of Angiotensin II in the regulation of sensory signaling, we characterized the AT1 expression in neuronal subpopulation of lower lumbar dorsal root ganglia under normal conditions and its alteration in neuropathic pain model. The characterization of AT1 expression was done under control and after the chronic constriction injury induced by four loose ligatures of the sciatic nerve representing the model of posttraumatic painful peripheral neuropathy. Major Angiotensin II receptor type was expressed in approximately 43 % of small-sized and 62 % of large-sized neurons in control. The AT1 overexpression after sciatic nerve ligation lasting 7 days was detected predominantly in small-sized AT1 immunoreactive neurons (about 38 % increase). Chronic constriction injury caused a statistically marked increase in number of the small-sized peptidergic (CGRP immunoreactive) neuronal subpopulation expressing AT1 (about 64 %). The subpopulations of AT1-immunoreactive and nonpeptide-containing primary sensory neurons revealed by IB4 binding, tyrosine hydroxylase- and parvalbumin-immunoreactive neurons were not markedly changed. Our results indicate that: (1) the AT1 overexpression after the chronic constriction injury is an important factor in Angiotensin II-potentiated pain perception; (2) Angiotensin II is involved in pathological mechanisms of neuropathic pain and this effect can be mediated perhaps in combination with other neuropeptides synthesized in the primary sensory neurons.

In addition to behavioral testing, the efficacy of neuroprotective therapies applied after spinal cord injury (SCI) is commonly evaluated by means of histological quantification of spared neural tissue. The primary insult itself, but mainly the pathological processes of secondary injury are the underlying causes of spinal tissue degeneration, the extent of which depends on the injury severity and post-injury time. Under-estimation of tissue loss due to spinal cord shrinkage and subjective evaluation (impeding reproducibility) are substantial factors that negatively affect the final results. Moreover, processing large numbers of stained spinal cord sections is very time-consuming. To overcome the problem, our new quantification approach combines a modified method for predicting the cross-sectional area at the lesion site with semi-automatic measurement of spared neural tissue and cystic cavities, using freely accessible National Institutes of Health (NIH) ImageJ software, with a Java-based image processing program. Based on the histological parameters measured after differing compression-induced SCI and correlated with behavioral outcomes, we can conclude that our new method is relatively fast, accurate, and optimally reproducible.

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.

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 with DMEM/F12 medium. It was demonstrated that the tissue preservation in the cranial and caudal segments immediately adjacent to the lesion, and neurofilament protection in the lateral columns may be essential for modulation of the key spinal microcircuits leading to a functional outcome. Tissue sparing observed only in the caudal sections, even though significant, was not sufficient for functional improvement in the 15N-SCI model.

Guanylyl cyclase (GC) as the effector molecule for nitric oxide (NO) plays a key role in the NO/cGMP signalling cascade. Based on these observations, our study focused on NO/sGC signalization in the bulbospinal respiratory pathway. The distribution of neuronal nitric oxide synthase (nNOS), β1 subunit of soluble guanylyl cyclase (β1sGC) and synaptophysin (SYN) was explored in the upper part of the respiratory pathway after C2-C3 hemisection of the spinal cord in male Wistar rats. Unilateral injection of Fluorogold into the phrenic nucleus (PN) at C4 level and survival of animals for 2 days revealed many Fluorogold fluorescent neurons in the ventral respiratory group (VRG) of the medulla, mostly on the contralateral side. Under physiological conditions we noted nNOS-fluorescent terminals of VRG neurons around β1sGC fluorescent motoneurons in the PN. A strong depletion of nNOS/SYN fluorescent terminals was noted 8 days after hemisection around alpha motoneurons in the PN on the contralateral side. On the side of injury, nNOS/SYN fluorescent puncta were detected around phrenic motoneurons only sporadically. Phrenic alpha motoneurons responded to C2-C3 hemisection by a loss of β1sGC positivity. The results confirm, that β1sGC immunoreactive phrenic motoneurons are innervated by nNOS positive terminals coming from the VRG.

Inhibition of angiotensin II receptor type 1 (AT1) reduces chronic inflammation associated with hypertension. We asked whether AT1 receptor inhibition would reduce the innate inflammatory response induced by bacterial lipopolysaccharide (LPS).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 mumol/l candesartan, an AT1 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.Human monocytes did not express detectable AT1 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.We hypothesize that the anti-inflammatory effects of candesartan in these cells are likely mediated by mechanisms unrelated to AT1 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.

ANG II AT1 receptor blockade reduces inflammation in hypertension. To determine whether ANG II AT1 receptor blockers (ARBs) influence the innate immune inflammatory response in normotensive rats, we studied rat plasma and spleen after a 3-day subcutaneous pretreatment with the ARB candesartan followed by a single dose of the bacterial endotoxin LPS (50 μg/kg ip). Peripheral administration of LPS to rodents produced a generalized inflammatory response with increased release of TNF-α, IL-1β, and IL-6 into the circulation. Candesartan pretreatment reduced the LPS-induced release of TNF-α, IL-1β, and IL-6 into the circulation. The red pulp of rat spleen expressed large numbers of AT1 receptors and the LPS receptors Toll-like receptor 4 and CD14. Candesartan administration significantly blocked AT1 receptors. The ARB reduced the LPS-induced upregulation of CD14 gene expression; expression of TNF-α and IL-6 mRNA and protein; expression of IL-1β and IκB-α mRNA; COX-2 mRNA and protein expression and PGE2 concentration; inducible nitric oxide synthase (iNOS) gene and protein expression and iNOS activity; and Nox2 gene expression and 8-isoprostane levels. In addition, candesartan reduced the CD14 protein expression in saline- and LPS-treated rats. Our results suggest that AT1 receptors are essential for the development of the full innate immune response to bacterial endotoxin. The ARB decreased the general peripheral inflammatory reaction to LPS and partially decreased the inflammatory response in the spleen. An unrestricted innate immune response to the bacterial endotoxin may have deleterious effects for the organism and may lead to development of chronic inflammatory disease. We postulate that ARBs may have therapeutic effects on inflammatory conditions.

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.

The physiological actions of brain Angiotensin II AT(2) receptors and their relationship to Angiotensin II AT(1) receptors remain controversial. To further clarify their role, we determined to what extent systemic administration of an AT(2) receptor antagonist affected AT(2) receptor binding within the brain and the expression of AT(1) receptors. For this purpose, we subcutaneously administered the AT(2) receptor antagonist PD123319 (1 mg/kg/day) to adult male rats for two weeks via osmotic minipumps. We also studied the content of pituitary adrenocorticotropic hormone and vasopressin, representative of hypothalamic-pituitary-adrenal axis activation, and the tyrosine hydroxylase gene expression in the locus coeruleus as a measure of central norepinephrine function. We found significant decreases in AT(2) receptor binding in brain areas inside the blood brain barrier, the inferior olive and the locus coeruleus. AT(2) receptor blockade increased AT(1) receptor binding and mRNA expression not only in the subfornical organ and the median eminence, situated outside the blood brain barrier, but also in the hypothalamic paraventricular nucleus, located inside the blood brain barrier. These changes paralleled decreased expression of tyrosine hydroxylase mRNA in the locus coeruleus and decreased pituitary adrenocorticotropic and vasopressin content. Our results demonstrate that sustained peripheral administration of an AT(2) antagonist decreases binding to brain AT(2) receptors, indicating that this drug is a useful tool for the study of their central role. AT(2) receptor activity inhibition up-regulates AT(1) receptor expression in specific brain areas. Blockade of brain AT(2) receptors is compatible with enhanced hypothalamic-pituitary-adrenal axis and decreased central sympathetic system activity.

To clarify the role of Angiotensin II in the regulation of peripheral sensory and motor systems, we initiated a study of the expression, localization and transport of Angiotensin II receptor types in the rat sciatic nerve pathway, including L(4)-L(5) spinal cord segments, the corresponding dorsal root ganglia (DRGs) and the sciatic nerve. We used quantitative autoradiography for AT(1) and AT(2) receptors, and in situ hybridization to detect AT(1A), AT(1B) and AT(2) mRNAs. We found substantial expression and discrete localization of Angiotensin II AT(1) receptors, with much higher numbers in the grey than in the white matter. A very high AT(1) receptor expression was detected in the superficial dorsal horns and in neuronal clusters of the DRGs. Expression of AT(1A) mRNA was significantly higher than that of AT(1B). AT(1) receptor binding and AT(1A) and AT(1B) mRNAs were especially prominent in ventral horn motor neurons, and in the DRG neuronal cells. Unilateral dorsal rhizotomy significantly reduced AT(1) receptor binding in the ipsilateral side of the superficial dorsal horn, indicating that a substantial number of dorsal horn AT(1) receptors have their origin in the DRGs. After ligation of the sciatic nerve, there was a high accumulation of AT(1) receptors proximal to the ligature, a demonstration of anterograde receptor transport. We found inconsistent levels of AT(2) receptor binding and mRNA. Our results suggest multiple roles of Angiotensin II AT(1) receptors in the regulation of sensory and motor functions.

Peripheral administration of bacterial endotoxin [lipopolysaccharide (LPS)] to rodents produces an innate immune response and hypothalamic-pituitary-adrenal axis stimulation. Renin-angiotensin-aldosterone system inhibition by angiotensin II AT1 receptor blockade has antiinflammatory effects in the vasculature. We studied whether angiotensin II receptor blockers (ARBs) prevent the LPS response. We focused on the adrenal gland, one organ responsive to LPS and expressing a local renin-angiotensin-aldosterone system. LPS (50 μg/kg, ip) produced a generalized inflammatory response with increased release of TNF-α and IL-6 to the circulation, enhanced adrenal aldosterone synthesis and release, and enhanced adrenal cyclooxygenase-2, IL-6, and TNF-α gene expression. ACTH and corticosterone release were also increased by LPS. Pretreatment with the ARB candesartan (1 mg/kg·d, sc for 3 d before the LPS administration) decreased LPS-induced cytokine release to the circulation, adrenal aldosterone synthesis and release, and cyclooxygenase-2 and IL-6 gene expression. Candesartan did not prevent the LPS-induced ACTH and corticosterone release. Our results suggest that AT1 receptors are essential for the development of the full innate immune and stress responses to bacterial endotoxin. The ARB decreased the general peripheral inflammatory response to LPS, partially decreased the inflammatory response in the adrenal gland, prevented the release of the pro-inflammatory hormone aldosterone, and protected the antiinflammatory effects of glucocorticoid release. An unrestricted innate immune response to the bacterial endotoxin may have deleterious effects for the organism and may lead to development of chronic inflammatory disease. We postulate that the ARBs may have therapeutic effects on inflammatory conditions.

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.

To clarify the role of Angiotensin II in the regulation of sensory signaling, we characterized the AT

1. This study was performed to compare both the Ca(2+)-dependent nitric oxide synthase (NOS) activity and the neuronal nitric oxide synthase immunoreactivity (nNOS-IR) in the rabbit lumbosacral spinal cord after 15 min abdominal aorta occlusion (ischemia in vivo) and oxygen-glucose deprivation of the spinal cord slices for 45 and 60 min (ischemia in vitro). All ischemic periods were followed by 15, 30 and 60 min reoxygenation in vitro. 2. Catalytic nitric oxide synthase activity was determined by the conversion of (L)-[(14)C]arginine to (L)-[(14)C]citrulline. Neuronal nitric oxide synthase immunoreactivity in the spinal cord was detected by incubation of sections with polyclonal sheep-nNOS-primary antibody and biotinylated anti-sheep secondary antibody. 3. Our results show that ischemia in vivo and the oxygen-glucose deprivation of spinal cord slices in vitro result in a time-dependent loss of constitutive NOS activity with a partial restoration of enzyme activity during 15 and 45 min ischemia followed by 30 min of reoxygenation. A significant decrease of enzyme activity was found during 60 min ischemia alone, which persisted up to 1 h of oxygen-glucose restoration. The upregulation of neuronal nitric oxide synthase was observed in the ventral horn motoneurons after all ischemic periods. The remarkable changes in optical density of neuronal nitric oxide synthase immunoreactive motoneurons were observed after 45 and 60 min ischemia in vitro followed by 30 and 60 min reoxygenation. 4. Our results suggest that the oxygen-glucose deprivation followed by reoxygenation in the spinal cord is adequately sensitive to monitor ischemia/reperfusion changes. It seems that 15 min ischemia in vivo and 45 min ischemia in vitro cause reversible changes, while the decline of Ca(2+)-dependent nitric oxide synthase activity after 60 min ischemic insult suggests irreversible alterations.

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 microvascular endothelium. In SHR, (pro)renin receptor mRNA and AT 1 receptor mRNA and protein expression were higher, whereas Aogen, ACE mRNA and AT 2 receptor mRNA and protein expression were lower than in WKY rats. Candesartan treatment increased Aogen, ACE and AT 2 receptor in SHR, and increased ACE and decreased Aogen in WKY rats, without affecting the (pro)renin and AT 1 receptors. Conclusions— Increased (pro)renin and AT 1 receptor expression in SHR substantiates the importance of the local RAS overdrive in the cerebrovascular pathophysiology in hypertension. AT 1 receptor blockade and increased AT 2 receptor stimulation after administration of candesartan may contribute to the protection against brain ischemia and inflammation.

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.

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 V1, V3, or CRF1 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 AT1 receptor stimulation determines the response to stress. An orally active, non-peptidic Ang II AT1 receptor antagonist blocks CRF and AVP release during stress, prevents the sympathoadrenal and hormonal stress reaction, the cortical CRF1 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 AT1 receptor stimulation, and should be studied together. Selective, safe antagonists of Ang II AT1 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. Drug Dev. Res. 65:237–269, 2005. © Published 2005 Wiley-Liss, Inc.

Changes in the level of cyclic 3',5'-guanosine monophosphate (cGMP) were studied one day after a surgically induced spinal cord constriction performed at the Th7 segment level in the dorsal, lateral and ventral white matter columns and in the non-compartmentalized white matter of Th5-Th6 segments, i.e., above the site of the spinal cord constriction and in Th8-Th9 segments, located below the spinal cord constriction. The midthoracic spinal cord constriction caused a significant decrease in the level of cGMP in the ventral column of Th5-Th6 segments and a significant increase in the lateral column of Th8-Th9 segments. The level of cGMP in the dorsal column, located either rostrally or caudally to the site of the spinal cord injury, remained unchanged. In addition, no significant changes in the level of cGMP were found in the non-compartmentalized white matter of Th5-Th6 and Th8-Th9 segments in response to constriction of the Th7 segment.

The regional distribution of catalytic NOS activity was studied in the lumbosacral segments of the spinal cord of the rabbit during single (8-min), twice (8-, 8-min) and thrice repeated (8-, 8-, 9-min) sublethal ischemia followed each time by 1h of reperfusion. Single ischemia/reperfusion induced a significant increase of cNOS activity in almost all spinal cord regions, with the exception of non-significant increase in the dorsal horn. Sublethal ischemia repeated twice produced a significant decrease of enzyme activity in the intermediate zone and ventral horn and an increase in the white matter columns. Within thrice repeated ischemia, the activity of cNOS in the gray matter regions was similar to that found after a single ischemia/reperfusion. For all the animals subjected to single and twice repeated sublethal ischemic insults, there was no neurological impairment. Following thrice repeated ischemic insults, four out of five of the experimental animals recovered only partially and one was completely paraplegic. Our results do not indicate a cumulative effect of repeated sublethal ischemia on cNOS activity and, consequently, on NO production. The NO generated during thrice repeated ischemia/reperfusion appears to have a detrimental effect on the neurological outcome.

The distribution and changes of catalytic nitric oxid synthase (cNOS) activity in the dorsal, lateral and ventral white matter columns at midthoracic level of the rabbit's spinal cord were studied in a model of surgically-induced spinal cord constriction performed at Th7 segment level and compared with the occurrence of nicotinamide adenine dinucleotide phosphate diaphorase expressing and neuronal nitric oxide synthase immunoreactive axons in the white matter of the control thoracic segments. Segmental and white-column dependent differences of cNOS activity were found in the dorsal (141.5 ± 4.2 dpm/μm protein), lateral (87.3 ± 11.5 dpm/μm protein) and ventral (117.1 ± 7.6 dpm/μm protein) white matter columns in the Th5-Th6 segments and in the dorsal (103.3 ± 15.5 dpm/μm protein), lateral (54.9 ± 4.9 dpm/μm protein), and ventral (86.1 ± 6.8 dpm/μm protein) white matter columns in the Th8-Th9 segments. A surgically-induced constriction of Th7 segment caused a disproportionate response of cNOS activity in the rostrally (Th5-Th6) and caudally (Th8-Th9) located segments in both lateral and ventral white matter columns. While a statistically significant decrease of cNOS activity was detected above the constriction site in the ventral columns, a considerable, statistically significant increase of cNOS activity was noted in the white lateral columns below the site of constriction. It is reasoned that the changes of cNOS activity may have adverse effects on nitric oxide (NO) production in the white matter close to the site of constriction injury, thus broadening the scope of the secondary mechanisms that play a role in neuronal trauma.

The regional distribution of cyclic 3′,5′-guanosine monophosphate was studied in the lumbosacral segments of the spinal cord of the rabbit under physiological conditions and following brief repeated sublethal ischemic insults. While the basal cGMP level in the gray matter was about 0.120 nmol cGMP/mg wet. wt., the level of cGMP in non-compartmentalized white matter was about half of this value. The highest level of cGMP in the compartmentalized gray matter was found in the dorsal horns, about 0.180 nmol cGMP/mg wet. wt., whereas the level of cGMP was greatly reduced in the ventral horns, reaching one half of the previous value. Multiple sublethal ischemic insults, repeated at 1-h intervals, caused a statistically significant decrease of cGMP in all gray matter regions. While the post-ischemic and post-reperfusion level of cGMP in the dorsal horns remained relatively high in comparison with the intermediate zone and ventral horns, the changes of cGMP level detected in the white matter columns differed considerably and resulted in a statistically significant cGMP increase in the dorsal and ventral columns and, vice versa, a statistically significant decrease of cGMP was found in the lateral columns.

Background context The loss of descending control after spinal cord injury (SCI) and incessant stimulation of Ia monosynaptic pathway, carrying proprioceptive impulses from the muscles and tendons into the spinal cord, evoke exaggerated α-motoneuron activity leading to increased reflex response. Previous results from our laboratory have shown that Ia monosynaptic pathway is nitrergic. Purpose The aim of this study was to find out whether nitric oxide produced by neuronal nitric oxide synthase (nNOS) plays a role in setting the excitability of α-motoneurons after thoracic spinal cord transection. Study design We tested the hypothesis that the inhibition of nNOS in α-motoneurons after SCI could have a neuroprotective effect on reflex response. Methods Rats underwent spinal cord transection at Th10 level followed by 7, 10, and 14 days of survival. The animals were treated with Baclofen (a gamma aminobutyric acid B receptor agonist, 3 μg/two times per day/intrathecally) applied for 3 days from the seventh day after transection; N -nitro-l-arginine (NNLA) (nNOS blocator) applied for the first 3 days after injury (20 mg/kg per day, intramuscularly); NNLA and Baclofen; or NNLA (60 mg/kg/day, single dose) applied on the 10th day after transection. We detected the changes in the level of nNOS protein, nNOS messenger RNA, and nNOS immunoreactivity. To investigate the reflex response to heat-induced stimulus, tail-flick test was monitored in treated animals up to 16 days after SCI. Results Our data indicate that Baclofen therapy is more effective than the combined treatment with NNLA and Baclofen therapy. The single dose of NNLA (60 mg/kg) applied on the 10th day after SCI or Baclofen therapy reduced nNOS expression in α-motoneurons and suppressed symptoms of increased reflex activity. Conclusions The results clearly show that increased nNOS expression in α-motoneurons after SCI may be pharmacologically modifiable with Baclofen or bolus dose of nNOS blocker.

The interruption of supraspinal input to the spinal cord leads to motor dysfunction and the development of spasticity. Clinical studies have shown that Baclofen (a GABA B agonist), while effective in modulating spasticity is associated with side-effects and the development of tolerance. The aim of the present study was to assess if discontinued Baclofen treatment and its repeated application leads antispasticity effects, and whether such changes affect neuronal nitric oxide synthase (nNOS) in the brainstem, nNOS and parvalbumin (PV) in lumbar α-motoneurons and glial fibrillary acidic protein in the ventral horn of the spinal cord. Adult male Wistar rats were exposed to Th9 spinal cord transection. Baclofen (30 mg/b.w.) diluted in drinking water, was administered for 6 days, starting at week 1 after injury and then repeated till week 4 after injury. The behavior of the animals was tested (tail-flick test, BBB locomotor score) from 1 to 8 weeks. Our results clearly indicate the role of nitric oxide, produced by nNOS in the initiation and the maintenance of spasticity states 1, 6 and 8 weeks after spinal trauma. A considerable decrease of nNOS staining after Baclofen treatment correlates with improvement of motor dysfunction. The findings also show that parvalbumin and astrocytes participate in the regulation of ion concentrations in the sub-acute phase after the injury.

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 AT1 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 AT1 receptor activation is an important regulatory factor in neuropathic pain perception and plays an important role in the injury of large-sized primary afferent neurons and activation of SGCs elicited by the CCI.

Spinal cord ischemia belongs to serious and relatively frequent diseases of CNS. The aim of the present study was to find out the vulnerability of nitrergic neurons to 15 min transient spinal cord ischemia followed by 1 and 2 weeks of reperfusion. We studied neuronal nitric oxide synthase (nNOS) and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) in structural elements of lumbosacral spinal cord along its rostrocaudal axis. In addition, a neurological deficit of experimental animals was evaluated. Spinal cord ischemia, performed on the rabbit, was induced by abdominal aorta occlusion using Fogarty catheter introduced into the right femoral artery for a period of 15 min. After surgical intervention the animals survived for 7 and 14 days. nNOS-immunoreactivity (nNOS-IR) was measured by immunohistochemical and NADPHd-positivity by histochemical method, and both immunohistochemical and histochemical stainings were quantified by densitometric analyses. Neurological deficit was evaluated according Zivin′s criteria. The number of nNOS-IR and/or NADPH-d positive neurons and the density of neuropil were markedly increased in superficial dorsal horn (laminae I–III) after 15 min ischemia and 7 days of reperfusion. However, ischemia followed by longer time of survival (14 days) returned the number of nNOS-IR and NADPH-d positive neurons to control. In the pericentral region (lamina X) containing interneurons and crossing fibers of spinal tracts, than in lamina VII and in dorsomedial part of the ventral horn (lamina VIII) we recorded a decreased number of nNOS-IR and NADPH-d positive neurons after both ischemia/reperfusion periods. In the medial portion of lamina VII and dorsomedial part of the ventral horn (lamina VIII) we observed many necrotic loci. This area was the most sensitive to ischemia/reperfusion injury. Fifteen minute ischemia caused a marked deterioration of neurological function of hind limbs, often developing into paraplegia. A quantitative immunohistochemical and histochemical study have shown a strong vulnerability of nitrergic neurons in intermediate zone to transient spinal cord ischemia.

Using immunohistochemistry, we detected the expression of neuronal nitric oxide synthase (nNOS) in ventral medullary gigantocellular reticular nuclei and in the lumbosacral spinal cord 10 days after thoracic transection in experimental rabbits. We tried to determine whether neurons located below the site of injury are protected by the calcium binding protein parvalbumin (PV). Changes of nNOS immunoreactivity (IR) in spinal cord were correlated with the level of nNOS protein in dorsal and ventral horns. Ten days after transection, nNOS was upregulated predominantly in lateral gigantocellular nuclei. In the spinal cord, we revealed a significant increase of nNOS protein in the dorsal horn. This is consistent with a higher density of punctate and fiber-like immunostaining for nNOS in laminae III–IV and the up-regulation of nNOS-IR in neurons of the deep dorsal horn. After surgery, the perikarya of motoneurons remained nNOS immunonegative. Contrary to nNOS, the PV-IR was upregulated in α-motoneurons and small-sized neurons of the ventral horn. However, its expression was considerably reduced in neurons of the deep dorsal horn. The findings indicate that spinal transection affects nNOS and PV in different neuronal circuits.

A prolonged exposure to vibration stimuli triggers pathological changes with many later manifested symptoms. Early vibration-induced changes are still not very well explored. Therefore, short 30 min vibration period per day with frequency 60 Hz repeated for 10 days was used, and the retrograde axonal transport from the sciatic nerve, the expression of calcitonin gene-related peptide (CGRP) and parvalbumin (PV) were studied in the dorsal root ganglia (DRGs) corresponding to lower lumbar spinal levels. Repeated vibration markedly decreased (25 and 34%) the accumulation of retrogradely transported Fluorogold to spinal motor neurons, whereas a significant increase (35 and 25%) was seen in the DRG primary sensory neurons corresponding to the L4 and L5 spinal level. Immunohistochemical studies showed a significant reduction of CGRP-positive small-sized neuronal cells in both DRGs. Fluoro-Jade labeling revealed that marked loss of CGRP-imunoreactive DRG sensory neurons is not due to neuronal degeneration. CGRP protein expression determined by Western blot analysis and optical density measurement, and NGF level measured by ELISA have been decreased, markedly only at the L4 DRG. PV protein expression was not affected by short repeated vibrations. Our results indicate that (a) short-lasting repeated vibrations affect the retrograde axonal transport in the DRG sensory neurons differently than in spinal motor neurons; (b) a decreased NGF-dependent CGRP production in the DRG primary sensory neurons plays an important role in early vibration-induced pathological mechanisms.

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 function upregulates AT(1) receptor transcription and expression in a tissue-specific manner and in cells not expressing AT(2) receptors. AT(1) upregulation explains the increased sensitivity to Angiotensin II characteristic of the AT(2)(-/-) phenotype, consistent with enhanced AT(1) receptor activation in a number of tissues.

In addition to regulating blood pressure, angiotensin II (Ang 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 Ang 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.

Systemic lipopolysaccharide (LPS) administration induces an innate immune response and stimulates the hypothalamic-pituitary-adrenal axis. We studied Angiotensin II AT(1) receptor participation in the LPS effects with focus on the pituitary gland. LPS (50 microg/kg, i.p.) enhanced, 3h after administration, gene expression of pituitary CD14 and that of Angiotensin II AT(1A) receptors in pituitary and hypothalamic paraventricular nucleus (PVN); stimulated ACTH and corticosterone release; decreased pituitary CRF(1) receptor mRNA and increased all plasma and pituitary pro-inflammatory factors studied. The AT(1) receptor blocker (ARB) candesartan (1mg/kg/day, s.c. daily for 3 days before LPS) blocked pituitary and PVN AT(1) receptors, inhibited LPS-induced ACTH but not corticosterone secretion and decreased LPS-induced release of TNF-alpha, IL-1beta and IL-6 to the circulation. The ARB reduced LPS-induced pituitary gene expression of IL-6, LIF, iNOS, COX-2 and IkappaB-alpha; and prevented LPS-induced increase of nNOS/eNOS activity. The ARB did not affect LPS-induced TNF-alpha and IL-1beta gene expression, IL-6 or IL-1beta protein content or LPS-induced decrease of CRF(1) receptors. When administered alone, the ARB increased basal plasma corticosterone levels and basal PGE(2) mRNA in pituitary. Our results demonstrate that the pituitary gland is a target for systemically administered LPS. AT(1) receptor activity is necessary for the complete pituitary response to LPS and is limited to specific pro-inflammatory pathways. There is a complementary and complex influence of the PVN and circulating cytokines on the initial pituitary response to LPS. Our findings support the proposal that ARBs may be considered for the treatment of inflammatory conditions.

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.

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 hours after nerve ligation there was no change in the patches or hot spots, but luminal AT(1)-reactivity 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.

Angiotensin II can promote cell stress, and the expression of its AT1 receptor is characteristic of neuronal populations that die off in multiple systems atrophy and Parkinson's disease. To explore the possible significance of these facts, we undertook to: (1) clarify the distribution of AT(1) in rat neurons; (2) use selective antagonists as a means of determining whether AT1 activation predisposes stressed neurons to die.AT1-expression was examined by immunohistochemistry and by autoradiography for [125I]-sarcosine1-angiotensin II binding in sensory, motor and autonomic neurons. To induce cell loss in a specific neuronal population, rats were given systemic i.v. injection of anti-acetylcholinesterase antibodies, which cause a delayed death of pre-ganglionic sympathetic neurons in the intermediolateral nucleus (IML). As pharmacologic intervention, some immunolesioned rats were treated with the selective AT1 antagonist, Candesartan.Immunohistochemistry and autoradiography revealed AT1 expression in dorsal root ganglia, superior cervical ganglion. In the dorsal horn of the spinal cord, AT1 immunostainining and angiotensin binding were both prominent. In ventral horn and IML, immunoreactivity for AT1 and choline acetyltransferase co-localized in pre-ganglionic sympathetic and somatic motor neurons. Immunolesion caused over 50% loss of IML perikarya within 3 months. Concurrent treatment with the AT1 antagonist, Candesartan, did not affect the outcome.AT1 expression is surprisingly widespread in sensory, autonomic and somatic motor neurons of the rat. This expression may be important to the normal physiology of these systems. Present data, however, do not support the concept that AT1 activation contributes to the loss of autonomic neurons after axonal damage.

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.

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.

Long-term pretreatment with an angiotensin II AT1 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 AT1 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 AT1 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. AT1 receptor blockade prevented the isolation-induced increase in brain AT1 receptors and decrease in AT2 binding in the locus coeruleus. AT1 receptor antagonism also prevented the increase in tyrosine hydroxylase mRNA in the locus coeruleus. Pretreatment with the AT1 receptor antagonist completely prevented the decrease in cortical CRF1 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 CRF1 receptors and the GABA(A) complex as molecular mechanisms responsible for the anti-anxiety effect of centrally acting AT1 receptor antagonists. We propose that AT1 receptor antagonists can be considered as compounds with possible therapeutic anti-stress and anti-anxiety properties.

Using nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry and nitric oxide synthase (NOS) immunocytochemistry combined with radioassay of calcium-dependent NOS activity, we examined the occurrence of NADPHd staining and NOS immunoreactivity (NOS-IR) in the dorsal root ganglia (DRG) neurons, dorsal root afferents, and axons projecting via gracile fascicle to gracile nucleus 14 days after unilateral sciatic nerve transection in the rabbit. Mild to moderate NADPHd staining and NOS-IR appeared in a large number of small and medium-sized to large neurons in the ipsilateral L4-L6 DRG, accompanied by enhanced NOS-IR of thick myelinated fibers in the ipsilateral L4-L6 dorsal roots. A noticeable increase in the density of punctate NADPHd staining occurred throughout laminae I-IV in the ipsilateral medial part of the dorsal horn in L4-L6 segments. Concurrently, a statistically significant decrease in the number of small NADPHd-exhibiting neurons in laminae I-II and, in contrast to this, a statistically significant increase of medium-sized to large NADPHd-stained somata in the ipsilateral laminae III-VI of L4-L6 segments were found. A detailed compartmentalization of L4-L6 segments into gray and white matter regions disclosed substantially increased catalytic NOS activity and inducible NOS mRNA levels in the dorsal horn and dorsal column ipsilaterally to the peripheral injury. A noticeable increase in the number of thick myelinated NADPHd-exhibiting and NOS-IR axons was noted in the ipsilateral gracile fascicle, terminating in dense, punctate NADPHd staining in the neuropil of the gracile nucleus. These observations indicate that the de novo-synthesized NOS in the lesioned primary afferent neurons resulting after sciatic nerve transection may be involved in an increase in NADPHd staining and NOS-IR in the ipsilateral dorsal roots and dorsal horn of L4-L6 segments, whence NOS could be supplied to ascending axons of the gracile fascicle.

Histochemical, immunocytochemical and radioassay study was performed to detect the occurrence of NOS-immunoreactive primary trigeminal sensory somata in the trigeminal ganglion, including their fiber components. Spinal trigeminal tract and sensory trigeminal nuclei were studied using the same methods. It was found that more than 30% of all somata in the trigeminal ganglion are NOS immunoreactive. Corresponding fibers were detected in the spinal trigeminal tract. NOS immunoreactive fibers of three different categories could be followed to terminate in the sensory trigeminal nuclei. Data presented here confirm that trigeminal sensory system is richly endowed with NOS and that NO is used to communicate between the first and second-order trigeminal sensory neurons. Acknowledgements: Supported by VEGA Grant no. 2/3217/23PS9, STAA Grant no. 51-013002 and by NIH grants NS 32794 and NS 40386 to M.M.