Region-specific sensitivity of the spinal cord to ischemia/reperfusion: the dynamic of changes in catalytic NOS activity.

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.

Neurobiological effects of microwave exposure: a review focused on morphological findings in experimental animals.

The possible risk of electromagnetic radiation (EMR) for nervous system is regularly published from the middle of 20th century. Numbers of neurobiological studies demonstrate that various EMR frequencies induce changes in nervous tissue of experimental animals but the evidence for health effect of EMR to the nervous system remains uncertain. To solve the fundamental questions about possible health hazard of modern technologies, the main producers of EMR, further intensive experimental studies on animals are needed. This review, focused on morphological findings achieved in various experimental animals, demonstrates that blood-brain barrier is the most studied morpho-functional unit of CNS in experiments with EMR. The morphological findings in experimental animals, in many cases controversial, put some evidence on nervous tissue structural damage after the EMR exposure. In spite of numerous literary data a wide range of contemporary neuro-morphological methods waits to be utilized in the EMR experimental paradigm. Using these methods could play an important role in answering the question about possible adverse effects of microwaves on nervous system.

Neuronal nitric oxide synthase immunopositivity in motoneurons of the rabbit's spinal cord after transient ischemia/reperfusion injury.

1. Motoneurons in the spinal cord are especially vulnerable to ischemic injury and selectively destroyed after transient ischemia. To evaluate the role of nitric oxide (NO) in the pathophysiology of the spinal cord ischemia, the expression of neuronal nitric oxide synthase (nNOS) in the motoneurons of the lumbosacral spinal cord was examined in the rabbit model of transient abdominal aorta occlusion. 2. The aim of the present study was to find if there is any consensus between the duration of transient abdominal aorta occlusion, nNOS positivity of the motoneurons and neurological hind limb impairment. 3. According to the degree of neurological damage (i.e., from the group with almost no sign of damage to a group with fully developed paraplegia), the experimental animals were divided into three groups. The respective spinal cord segments of each experimental group were compared to the control group. 4. Spinal cord ischemia (15 min) was induced by Fogarty arterial embolectomy catheter occlusion of abdominal aorta with a reperfusion period of 7 days. On seventh day, the sections of lumbosacral segments were immunohistochemically treated and L1-L7, and S1-S2 segment sections were monitored using light microscopy.

Development of GABA-sensitive spasticity and rigidity in rats after transient spinal cord ischemia: a qualitative and quantitative electrophysiological and histopathological study.

Transient spinal cord ischemia may lead to a progressive degeneration of spinal interneurons and subsequently to increased hind limb motor tone. In the present work we sought to characterize the rigidity and spasticity components of this altered motor function by: i) tonic electromyographic activity measured in gastrocnemius muscle before and after ischemia, ii) measurement of muscle resistance during the period of ankle flexion and corresponding changes in electromyographic activity, iii) changes in Hoffmann reflex, and, iv) motor evoked potentials. In addition the effect of intrathecal treatment with baclofen (GABAB receptor agonist; 1 microg), nipecotic acid (GABA uptake inhibitor; 300 microg) and dorsal L2-L5 rhizotomy on spasticity and rigidity was studied. Finally, the changes in spinal choline acetyltransferase (ChAT) and vesicular glutamate transporter 2 and 1 (VGLUT2 and VGLUT1) expression were characterized using immunofluorescence and confocal microscopy. At 3-7 days after ischemia an increase in tonic electromyographic activity with a variable degree of rigidity was seen. In animals with modest rigidity a velocity-dependent increase in muscle resistance and corresponding appearance in electromyographic activity (consistent with the presence of spasticity) was measured during ankle rotation (4-612 degrees /s rotation). Measurement of the H-reflex revealed a significant increase in Hmax/Mmax ratio and a significant loss of rate-dependent inhibition. In the same animals a potent increase in motor evoked potential amplitudes was measured and this change correlated positively with the increased H-reflex responses. Spasticity and rigidity were consistently present for a minimum of 3 months after ischemia. Intrathecal treatment with baclofen (GABA B receptor agonist) and nipecotic acid (GABA uptake inhibitor) provided a significant suppression of spasticity, rigidity, H-reflex or motor evoked potentials. Dorsal L2-L5 rhizotomy significantly decreased muscle resistance but had no effect on increased amplitudes of motor evoked potentials. Confocal analysis of spinal cord sections at 8 weeks-12 months after ischemia revealed a continuing presence of ChAT positive alpha-motoneurons, Ia afferents and VGLUT2 and VGLUT1-positive terminals but a selective loss of small presumably inhibitory interneurons between laminae V-VII. These data demonstrate that brief transient spinal cord ischemia in rat leads to a consistent development of spasticity and rigidity. The lack of significant suppressive effect of dorsal L2-L5 rhizotomy on motor evoked potentials response indicates that descending motor input into alpha-motoneurons is independent on Ia afferent couplings and can independently contribute to increased alpha-motoneuronal excitability. The pharmacology of this effect emphasizes the potent role of GABAergic type B receptors in regulating both the spasticity and rigidity.

Spatiotemporal alterations of the NO/NOS neuronal pools following transient abdominal aorta occlusion: morphological and biochemical studies in the rabbit.

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.

Alterations of the expression and activity of midbrain nitric oxide synthase and soluble guanylyl cyclase in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsonism in mice.

The study was aimed at investigating the expression and the activity of neuronal nitric oxide synthase, and of soluble guanylyl cyclase and phosphodiesterase activities that regulate guanosine 3',5'-cyclic monophosphate level in the midbrain, in a mouse model of PD using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine injections. Adult male mice of the C57/BL strain were given three i.p. injections of physiological saline or three i.p. injections of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine solution in physiological saline at 2 h intervals (summary 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine dose: 40 mg/kg), and were killed 3, 7, or 14 days later. mRNA, protein level, and/or activities of neuronal nitric oxide synthase, soluble guanylyl cyclase, phosphodiesterase and guanosine 3',5'-cyclic monophosphate were determined. Immunohistochemistry showed about 75% decrease in the number of tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta. Mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine showed increased midbrain guanylyl cyclase and total nitric oxide synthase activities at 3, 7, and 14 days post-treatment. The specific neuronal nitric oxide synthase inhibitor 7-nitroindazole (10 microM) and the specific inducible nitric oxide synthase inhibitor 1400W (10 microM) inhibited the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced excess in nitric oxide synthase activity by 63-70 and 13-25%, respectively. The increases in total midbrain nitric oxide synthase activity were accompanied by elevated guanosine 3',5'-cyclic monophosphate, enhanced expression of neuronal nitric oxide synthase and of the beta1 subunit of guanylyl cyclase at both mRNA and protein levels that persisted up to the end of the observation period, and by enhanced neuronal nitric oxide synthase and guanylyl cyclase beta1 immunoreactivities in substantia nigra pars compacta 7 and 14 days after the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment. The increases in guanylyl cyclase activity were found to occur exclusively due to increased maximal enzyme activity. No 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced change in phosphodiesterase activity has been detected in any brain region studied. 7-Nitroindazole prevented a significant increase in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced midbrain guanosine 3',5'-cyclic monophosphate level and neurodegeneration of dopaminergic neurons. These results raise the possibility that the nitric oxide/guanylyl cyclase/guanosine 3',5'-cyclic monophosphate signaling pathway may play a role in maintaining dopaminergic neurons function in substantia nigra pars compacta.

Experimental cauda equina compression induces HSP70 synthesis in dog.

The heat shock protein 70 (HSP70) is a key component of the stress response induced by various noxious conditions such as heat, oxygen stress, trauma and infection. In present study we have assessed the consequences of the compression of lower lumbar and sacral nerve roots caused by a multiple cauda equina constrictions (MCEC) on HSP70 immunoreactivity (HSP70-IR) in the dog. Our data indicate that constriction of central processes evokes HSP70 up-regulation in the spinal cord (L7, S1-Co3) as well as in the corresponding dorsal root ganglion cells (DRGs) (L7-S1) two days following injury. A limited number of bipolar or triangular HSP-IR neurons were found in the lateral collateral pathway (LCP) as well as in the pericentral region (lamina X) of the spinal cord. In contrast, a high number of HSP70 exhibiting motoneurons with fine processes appeared in the ventral horn (laminae VIII-IX) of lumbosacral segments. Concomitantly, close to them a few lightly HSP70-positive neuronal somata or cell bodies lacking the HSP70-IR occurred. In the DRGs, HSP70 expression was mildly up-regulated in small and medium-sized neurons and in satellite cells. On the contrary, DRGs from intact or sham-operated dogs did not reveal HSP70 specific neuronal staining. In conclusion, we have demonstrated that the MCEC in dogs mimicking the cauda equina syndrome in clinical settings evokes expression of HSP70 synthesis in specific neurons of the lumbo-sacro-coccygeal spinal cord segments and in small and medium sized neurons of corresponding DRGs. This suggests that HSP70 may play an active role in neuroprotective processes partly by maintaining intracellular protein integrity and preventing the neuronal degeneration in this experimental paradigm.

Fluoro-Jade B evidence of induced ischemic tolerance in the rat spinal cord ischemia: physiological, neurological and histopathological consequences.

Fluoro-Jade B, a marker of degenerating neurons, was used to label histopathological changes in the rat spinal cord after transient ischemia and ischemic preconditioning (IPC). To characterize postischemic neurodegenerations and consequent neurological changes, a particular attention was paid to the standardization of ischemic conditions in animals of both groups. 1. The control ischemic rats were submitted to a reversible occlusion of descending aorta by insertion and subsequent inflation of a 2F Fogarty catheter for 12 min. 2. In the IPC rats, an episode of short 3 min occlusion and 30 min reperfusion preceded the 12 min ischemia. Postischemic motor function testing (ambulation and stepping) was provided repeatedly for evaluation of neurological status 2 h and 24 h after surgery and at the end of postischemic survival, i.e. after 48 h. Fluoro-Jade B staining was used to demonstrate degenerated neurons. In the control rats, neurological consequences of histopathological changes in lumbosacral spinal cord, manifested as paraplegia, were present after 12 min ischemia. Thus, numbers of degenerated Fluoro-Jade B positive cells were visible in gray matter of the most injured L(4)-S(2) spinal cord segments. Slight motor function impairment, consequential from significant decreasing in Fluoro-Jade B-positivity in the L(4)-S(2) spinal cord segments of the IPC rats, was considered the pathomorpfological evidence that IPC induces spinal cord tolerance to ischemia. Our results are consistent with the previously published silver impregnation method for histopathological demonstration of ischemic degeneration.

[Ischemic-reperfusion paraplegia in the dog and its light microscopy imaging]. / Ischemicko-reperfúzna paraplégia u psa a jej svetelnomikroskopický obraz.

Paraplegia, which develops after operation on aorta, represents a real catastrophe for the patient and for the surgeon. The aim of the present work was to investigate the light microscopy picture of this complication and consequently better understand related processes. Twenty one adult dogs, cross breeds of both sexes, weight 18-25 kg, were divided into four groups: 1. Controls (n = 3); 2.30-min ischemia induced by occlusion of thoracic aorta by a tourniquet, followed for 30 min survival (n = 6); 3.30-min ischemia and 72 h of survival (n = 6); 4) 30-min ischemia and 6 days of survival (n = 6). All these manipulations were made in sterile conditions under general anesthesia. As soon as the planned time of survival passed, the animals were flushed out, in deep pentobarbital anesthesia, with 3,000 ml of sodium chloride and fixed with 3,000 ml of 10% neutral formaldehyde. Sections, 30 microns thick, from L3-S1 medulla segments were processed in the laboratory of Neurobiological Institute by the method of Nauta for light microscopy examination. Neurohistological picture was characterized by a marked damage of the medulla neurons. The changes proved to be irreversible and resulted, in the course of six days of survival, to death of the cells, characterized by their disintegration. The results indicate that the only rational procedure in conditions of threatening ischemic-reperfusion injury of medulla is to prevent it.

Cauda equina syndrome and nitric oxide synthase immunoreactivity in the spinal cord of the dog.

The development of the cauda equina syndrome in the dog and the involvement of spinal nitric oxide synthase immunoreactivity (NOS-IR) and catalytic nitric oxide synthase (cNOS) activity were studied in a pain model caused by multiple cauda equina constrictions. Increased NOS-IR was found two days post-constriction in neurons of the deep dorsal horn and in large, mostly bipolar neurons located in the internal basal nucleus of Cajal seen along the medial border of the dorsal horn. Concomitantly, NOS-IR was detected in small neurons close to the medioventral border of the ventral horn. High NOS-IR appeared in a dense sacral vascular body close to the Lissauer tract in S1-S3 segments. Somatic and fiber-like NOS-IR appeared at five days post-constriction in the Lissauer tract and in the lateral and medial collateral pathways arising from the Lissauer tract. Both pathways were accompanied by a dense punctate NOS immunopositive staining. Simultaneously, the internal basal nucleus of Cajal and neuropil of this nucleus exhibited high NOS-IR. A significant decrease in the number of small NOS immunoreactive somata was noted in laminae I-II of L6-S2 segments at five days post-constriction while, at the same time, the number of NOS immunoreactive neurons located in laminae VIII and IX was significantly increased. Moreover, high immunopositivity in the sacral vascular body persisted along with a highly expressed NOS-IR staining of vessels supplying the dorsal sacral gray commissure and dorsal horn in S1-S3 segments. cNOS activity, based on a radioassay of compartmentalized gray and white matter regions of lower lumbar segments and non-compartmentalized gray and white matter of S1-S3 segments, proved to be highly variable for both post-constriction periods.

The effect of repeated sublethal ischemia on NO/cGMP signal transduction system in gray matter of the rabbit spinal cord.

BACKGROUND: The activation of the soluble guanylate cyclase and the production of cyclic 3',5'-guanosine monophosphate (cGMP) have been reported as the primary cellular response to nitric oxide (NO) in the nervous system. Previous results indicated, that three-fold sublethal ischemia repeated at 1-h intervals induces damage in gray matter of the spinal cord. However, little is known about the changes of NO/cGMP signal transduction system in gray matter of the spinal cord under conditions of repeated sublethal ischemia. MAIN PURPOSE: The aim of this study was to compare the catalytic NOS activity and cGMP concentration in the gray matter regions of lumbosacral spinal cord segments (dorsal horn, zona intermedia, ventral horn) after three (8-, 8-, 9-min) sublethal occlusions repeated at 1-h intervals. METHODS: Twenty male rabbits, weighing 2.5-3.5 kg were used in the experiments. They were divided into two experimental groups: (1) control animals (n = 10); (2) animals subjected to three brief (8-, 8-, 9-min) occlusions, each time repeated by reperfusion lasting for 1 h (n = 10). Ischemia of lumbosacral segments was induced by ligation of the abdominal aorta just below the left renal artery (DeGirolami and Zivin, 1982). The catalytic NOS activity was determined by conversion of [3H]-L-arginine to [3H]-L-citrulline according to the method of Bredt and Snyder (1990) slightly modified by Strosznajder and Chalimoniuk (1996). cGMP concentration was assessed by radioimmunoassay method (RIA). RESULTS: Repeated sublethal ischemia evoked a slight increase in catalytic NOS activity over control values in all gray matter regions. On the other hand, cGMP concentration in gray matter regions has a decreasing character, in a descending order: dorsal horn > zona intermedia > ventral horn. A significant impairment of NO-cGMP signal transduction was detected in the intermediate zone and ventral horns. CONCLUSIONS: Our results indicate that threefold (8-, 8-, 9-min) sublethal ischemia repeated in 1 h intervals of reperfusion causes the impairment of NO/cGMP signal transduction system in gray matter of lumbosacral spinal cord segments and the extent of impairment is region-specific. This finding correlates with the neurological hindlimbs impairment in experimental animals. (Tab. 1, Fig. 2, Ref. 39.)

The regional changes of the catalytic NOS activity in the spinal cord of the rabbit after repeated sublethal ischemia.

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 1 h 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.

Effect of spinal cord compression on cyclic 3',5'-guanosine monophosphate in the white matter columns of rabbit.

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.

Cauda equina syndrome.

Single or double-level compression of the lumbosacral nerve roots located in the dural sac results in a polyradicular symptomatology clinically diagnosed as cauda equina syndrome. The cauda equina nerve roots provide the sensory and motor innervation of most of the lower extremities, the pelvic floor and the sphincters. Therefore, in a fully developed cauda equina syndrome, multiple signs of sensory disorders may appear. These disorders include low-back pain, saddle anesthesia, bilateral sciatica, then motor weakness of the lower extremities or chronic paraplegia and, bladder dysfunction. Multiple etiologies can cause the cauda equina syndrome. Among them, non-neoplastic compressive etiologies such as herniated lumbosacral discs and spinal stenosis and spinal neoplasms play a significant role in the development of the cauda equina syndrome. Non-compressive etiologies of the cauda equina syndrome include ischemic insults, inflammatory conditions, spinal arachnoiditis and other infectious etiologies. The use of canine, porcine and rat models mimicking the cauda equina syndrome enabled discovery of the effects of the compression on nerve root neural and vascular anatomy, the impairment of impulse propagation and the changes of the neurotransmitters in the spinal cord after compression of cauda equina. The involvement of intrinsic spinal cord neurons in the compression-induced cauda equina syndrome includes anterograde, retrograde and transneuronal degeneration in the lumbosacral segments. Prominent changes of NADPH diaphorase exhibiting, Fos-like immunoreactive and heat shock protein HSP72 were detected in the lumbosacral segments in a short-and long-lasting compression of the cauda equina in the dog. Developments in the diagnosis and treatment of patients with back pain, sciatica and with a herniated lumbar disc are mentioned, including many treatment options available.

Fos protein expression in sacral spinal cord in relation to early phase of cauda equina syndrome in dogs.

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

Regional changes of cyclic 3',5'-guanosine monophosphate in the spinal cord of the rabbit following brief repeated ischemic insults.

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.

Effect of midthoracic spinal cord constriction on catalytic nitric oxide synthase activity in the white matter columns of rabbit.

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/microm protein), lateral (87.3 +/- 11.5 dpm/microm protein) and ventral (117.1 +/- 7.6 dpm/microm protein) white matter columns in the Th5-Th6 segments and in the dorsal (103.3 +/- 15.5 dpm/microm protein), lateral (54.9 +/- 4.9 dpm/microm protein), and ventral (86.1 +/- 6.8 dpm/microm 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.

Short-term changes of NADPH diaphorase-exhibiting neuronal pools in the spinal cord of rabbit after repeated sublethal ischemia.

The aim of this study was the histochemical characterization of NADPH diaphorase-positive neuronal pools in the rabbit lumbosacral segments using a model of single, repeated and multiple sublethal spinal cord ischemia. Following a single 8-min sublethal spinal cord ischemia and 1-hour reperfusion, the staining of NADPH diaphorase-exhibiting neurons in the dorsal horn, pericentral region, dorsal gray commissure and sacral parasympathetic nucleus was comparable with the control sections. In contrast to the foregoing sublethal ischemia, a regionally different somatic NADPH diaphorase (NADPHd) staining was found after multiple sublethal spinal cord ischemia. Whereas an almost complete loss of the staining of large NADPHd-exhibiting somata in the pericentral region was detected, the staining of the NADPHd-exhibiting neuronal pools in the deep dorsal horn and sacral parasympathetic nucleus was fully preserved. Concomitantly, a prominent reduction of small NADPH diaphorase-positive neurons was noted in the superficial dorsal horn layers of lower lumbar and sacral segments.

Incipient cauda equina syndrome as a model of somatovisceral pain in dogs: spinal cord structures involved as revealed by the expression of c-fos and NADPH diaphorase activity.

Segmental and laminar distribution of Fos-like immunoreactive, reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd)-exhibiting and double-labeled (Fos-like immunoreactive and NADPHd-exhibiting) neurons was examined in lower lumbar and sacral segments of the dog spinal cord using the model of multiple cauda equina constrictions. NADPHd histochemistry was used as marker of nitric oxide synthase-containing neurons. The appearance and the time-course of Fos-like immunoreactive, NADPHd and double-labeled neurons was studied at 2 h and 8 h postconstriction characterized as the incipient phase of cauda equina syndrome. The occurrence of Fos-like immunoreactive and NADPHd-exhibiting neurons in fully developed cauda equina syndrome was studied at five days postconstriction. An increase in Fos-like immunoreactivity in superficial laminae (I-II) and an enhanced NADPHd staining of lamina VIII neurons were found. A statistically significant increase in Fos-like immunoreactive neurons was found in laminae I-II and VIII-X 8 h postconstriction, and in contrast, a prominent decrease in Fos-like immunoreactive neurons was found in laminae I-II, accompanied by a statistically significant increase in Fos-like immunoreactive neurons in more ventrally located laminae VII-X at five days postconstriction. Quantitative analysis of laminar distribution of constriction-induced NADPHd-exhibiting neurons revealed a considerable increase in these neurons in laminae VIII-IX 8 h postconstriction and a statistically highly significant increase in NADPHd-exhibiting neurons in laminae VII-X five days postconstriction. Concurrently, the number of NADPHd-exhibiting neurons in laminae I-II was greatly reduced. While a low number of double-labeled neurons was found throughout the gray matter of lower lumbar and sacral segments at 2 h postconstriction, a statistically significant number of double-labeled neurons was found in lamina X 8 h and in laminae VII-X five days postconstriction. The course and distribution of anterograde degeneration resulting five days after multiple cauda equina constrictions are compared with segmental and laminar distribution of Fos-like immunoreactive and NADPHd-exhibiting neurons. Prominent involvement of the spinal cord neurons appearing in the lumbosacral segments at the early beginning and in fully developed cauda equina syndrome results in a Fos-like immunoreactivity and strongly enhanced NADPHd staining of some neuronal pools. Under such circumstances, an early cauda equina decompression surgery is advisable aimed at decreasing or preventing the derangement of the neural circuits in the lumbosacral segments.

Segmental and laminar distributions of nicotinamide adenine dinucleotide phosphate-diaphorase-expressing and neuronal nitric oxide synthase-immunoreactive neurons versus radioassay detection of catalytic nitric oxide synthase activity in the rabbit spinal cord.

The distributions of neuronal nitric oxide synthase-immunoreactive neurons and of nicotinamide adenine dinucleotide phosphate-diaphorase activity were studied in the C6, Th2, L1, L5, S2 and S3 segments and laminae in the rabbit spinal cord and compared with the catalytic nitric oxide synthase activity, determined by monitoring the conversion of [3H]arginine to [3H]citrulline in the same segments and laminae. Morphologically, a heterogeneous population of nicotinamide adenine dinucleotide phosphate-diaphorase-expressing and neuronal nitric oxide synthase-immunoreactive neurons was detected in the superficial and deep dorsal horn and the pericentral region in all segments studied, and in the intermediolateral cell column of the thoracic and lumbosacral segments. A disproportionate distribution of both neuronal categories which had a significantly higher number of nicotinamide adenine dinucleotide phosphate-diaphorase-expressing rather than neuronal nitric oxide synthase-immunoreactive cell bodies was found in all segments. The catalytic nitric oxide synthase activity was distributed unequally in the C6, Th2, L1, L5, S2 and S3 segments, with a comparatively low value in the Th2 segment (70 +/- 5.1 d.p.m./microg protein) in comparison with the S3 segment, where the highest level (140 +/- 5.5 d.p.m./microg protein) was found. A close correlation between the number of neuronal nitric oxide synthase-immunoreactive somata and catalytic nitric oxide synthase activity was revealed in the dorsal horn (laminae I-VI). Whereas a low number of neuronal nitric oxide synthase-immunoreactive somata in laminae VII-X was found in the L5, S2 and S3 segments, the values of catalytic nitric oxide synthase activity in the same laminae and segments were found to be exceedingly high. These findings indicate that the occurrence of many neuronal nitric oxide synthase-immunoreactive fibers (mainly axons), and dense, punctate, non-somatic neuronal nitric oxide synthase immunopositivity in the neuropil staining of the same laminae and segments, can substantially enhance catalytic nitric oxide synthase activity.