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.

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.

[Changes in phospholipids after repeated induced sublethal spinal cord ischemia in rabbits]. / Zmeny fosfolipidov po opakovanom navodení subletálnej ischémie miechy králika.

BACKGROUND: Degradation of membrane bound phospholipids in CNS during ischaemia begins with extreme rapidity. Sublethal ischaemia influences ischaemic tolerance in the affected neurons and is stressful enough to induce neuronal changes such as postischaemic hypoperfusion, transient suppression of protein synthesis and induction of stress (HSP) proteins. It seems, that the nature of factors responsible for ischaemic tolerance may involve the activation of multiple different systems. MAIN PURPOSE: The aim of this study was to investigate the changes of phospholipids in gray matter regions of spinal cord following sublethal ischaemia repeated in long intervals of reperfusion. METHODS: Male rabbits, weight range 2.5-3.5 kg were used in the experiment. They were divided in following groups : 1. control animals; 2. animals subjected to 25 min ischaemia; 3. animals subjected to 25 min ischaemia and 3 h of reperfusion; 4. animals subjected to sublethal (8-8-9 min) ischaemia repeated in long-lasting (8-8-24 h) intervals of reperfusion. Phospholipids were separated by thin layer chromatography, lipidic phosphorus was assessed spectrophotometrically. RESULTS: Sublethal ischaemia repeated in long-lasting intervals of reperfusion increased the concentration of phospholipids to control levels in all gray matter regions. The resynthesis in the dorsal horns, of PC and PE in the ventral horns and of PC in the intermediate zone. CONCLUSIONS: An excessive renewal of phospholipids after sublethal ischaemia repeated in longer intervals of reperfusion was most pronounced in the eh dorsal horns of the spinal cord and can be the result of many defensive cellular mechanisms.

Phospholipid composition in spinal cord regions after ischemia/reperfusion.

Ischemia-reperfusion induced changes in concentration of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI) and sphingomyelin (SM) in the gray matter taken in toto, white matter, dorsal horns, intermediate zone and ventral horns of the rabbit's spinal cord were studied and compared with neurohistopathological changes. With the exception of PI concentration in the dorsal horns, ischemia of 25 min caused significant degradation of all phospholipids. While short-lasting recirculation (1 h) did not returned the levels of phospholipids to control values, postischemic recirculation for 3 h sharply increased the resynthesis of all phospholipids, but only the concentration of PE, PS, and PI in the dorsal horns and PC in the intermediate zone significantly improved and returned close to control values. Corresponding neurohistopathological changes resulting after the same reperfusion periods are given.

Reduced nicotinamide adenine dinucleotide phosphate diaphorase in the spinal cord of dogs.

The distribution of somatic, fibre-like and punctate, non-somatic reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity was examined in dog spinal cord using horizontal, sagittal and transverse sections. The morphological features of NADPH diaphorase exhibiting neurons divided into six different neuronal types (N1-N6) were described and their laminar distribution specified. Major cell groups were identified in the superficial dorsal horn and around the central canal at all spinal levels, and in the intermediolateral cell column at thoracic level. NADPH diaphorase exhibiting neurons of the pericentral region were distributed in a thin subependymal cell column containing longitudinally-arranged small bipolar neurons with processes penetrating deeply into the intermediolateral cell column and/or running rostrocaudally in the subependymal layer. The second pericentral cell column located more laterally in lamina X contains large, intensely-stained NADPH diaphorase exhibiting neurons with long dendrites radiating in the transverse plane. Neurons of the sacral parasympathetic nucleus seen in segments S1-S3 exhibited prominent NADPH diaphorase activity accompanied by heavily-stained fibres extending from Lissauer's tract through lamina I along the lateral edge of the dorsal horn to lamina V. A massive dorsal gray commissure, with high NADPH diaphorase activity, was found in segments S1-S3. At the same segmental level a prominent group of moderately-stained motoneurons was detected in the dorsolateral portion of the anterior horn. Fibre-like NADPH diaphorase activity was found in the superficial dorsal horn and pericentral region in all segments studied. Punctate, non-somatic NADPH diaphorase activity was detected in the superficial dorsal horn, in the pericentral region all along the rostrocaudal axis and in the nucleus phrenicus (segments C4-C5), nucleus dorsalis (segments Th2-L2), nucleus Y (segments S1-S3), and the dorsal part of the dorsal gray commissure (S1-S3). A schematic diagram documenting the segmental and laminar distribution of NADPH diaphorase activity is given.

Regional changes of membrane phospholipid concentrations in rabbit spinal cord following brief repeated ischemic insults.

Changes in the concentration of membrane-bound phospholipids following single (25-min) spinal cord ischemia and 3 h of reperfusion were determined. These were compared with the changes following brief repeated (8-, 8-, and 9-min) ischemia followed each time by reperfusion for 1 h, or the same periods of ischemia followed by 8 h, 8 h, and 24 h of reperfusion, respectively. Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and sphingomyelin (SM) were assayed in regions of the spinal cord of the rabbit, including gray matter, white matter, dorsal horns, intermediate zone, and ventral horns. The brief repeated ischemia with 1-h reperfusions produced more extensive degradation of phospholipids in almost all regions compared with the equivalent time of ischemia (25 min) in a single period. After a lengthy reperfusion after repeated ischemia, the phospholipids were resynthesized with the exception of the phosphatidylinositol in the gray matter. The resynthesis was most pronounced in the dorsal horns and in the white matter.

Postischemic reperfusion causes a massive calcium overload in the myelinated spinal cord fibers.

The visualization of Ca binding in the myelinated axons of lumbosacral segments of rabbit was done at the electron microscopic level using the spinal cord ischemia model. To assess the calcium accumulation, the binding agent pyroantimonate was used. Nonsignificant Ca2+ binding was found in the myelinated axons after 40 min of ischemia followed immediately by perfusion fixation. A high concentration of calcium pyroantimonate deposits, seen as electron dense particles, was detected in the myelin interlamellar clefts and axoplasm. The paranodal region was the most affected site.

Multiple protracted cauda equina constrictions cause deep derangement in the lumbosacral spinal cord circuitry in the dog.

Neuropathological changes of the neuronal pools and spinal cord circuitry in the lumbosacral segments were studied in a canine model of multiple protracted cauda equina constrictions. Anterograde degeneration of all sacrococcygeal and L7 dorsal root fibers was detected in S1-S3 and lower lumbar segments. A narrow degenerated gracile fascicle was found in all thoracic and cervical segments terminating in the gracile nucleus. Transneuronal degeneration of middle-sized and large neurons, located in S1-S3 and sporadically in L7 segments, was noted. Identical transneuronal degeneration was seen in a group of small neurons located in the ventralmost part of lamina VII in S1-S3 segments. Simultaneously, a terminal degeneration was detected in the lateral cervical nucleus and in the ventral posterior lateral nucleus of the ventrobasal thalamic complex. Concomitantly, a fully developed retrograde degeneration affecting motoneurons in the ventrolateral portion of the anterior horn in S1-S3 segments appeared.

Early neurohistopathological changes of canine lumbosacral spinal cord segments in ischemia-reperfusion-induced paraplegia.

Mapping the canine lumbosacral spinal cord neurons damaged by ischemia-reperfusion after high thoracic aorta ligation was performed using the Nauta degenerating method. Highly Nauta-positive perikarya of the long ascending projection systems in the 4th to 6th dorsal layer, interneurons in the 7th layer and motoneurons in the 8th and 9th layers in L3-S3 segments subjected to 30 min of ischemia and 30 min of reperfusion were localized and their laminar distribution was specified. Spastic paraplegia fully developed 2 days postoperatively after 30 min of aortic ligation is neurohistopathologically characterized by occurrence of enlarged Nauta-positive boutons with prevailing localization in the 4th to 8th layer of the gray matter.

[Effect of ischemia and recirculation on the ultrastructure of the ventral horns of the rabbit spinal cord and accumulation of calcium in motoneurons]. / Vliianie ishemii i retsirkuliatsii na ul'trastrukturu ventral'nykh rogov spinnogo mozga krolika i akkumuliatsiiu kal'tsiia v motoneironakh.

The effect of 40-min ligation of the abdominal aorta and 60-min recirculation was investigated with the use of pyroantimonate. All the motoneurons were markedly damaged. Vacuolized mitochondria and basal membrane of capillaries contained calcium deposits.

Spinal ascending projections to the nucleus tractus spinalis nervi trigemini of the dog.

Seven dogs were subjected 30 min to ligation of the thoracic aorta and were then kept alive 6-7 days after the ligature had been removed. Their spinal cord and brain stem were treated by the Nauta-Gygax method and the extent and appearance of preterminal and terminal degeneration of certain ascending spinal systems were analysed. In the medulla oblongata region, marked degenerating fibres from the lower thoracic and lumbosacral cord segments were found in the nucleus tractus spinalis nervi trigemini. Preterminal and terminal degenerating fibres were visualized in the caudal part of the trigeminal nuclear complex. Comparison with the literature showed these to be previously unknown projections with a relationship to the nucleus tractus spinalis nervi trigemini.

Nucleus ultrastructure of dog dorsal root ganglia cells after partial ischemia.

The partial ischemia of the adult dogs, both males and females was induced by the ligature of aorta abdominalis above the branching of arteria coeliaca within 80 minutes and 120 minutes duration with the following preparation of L6 und L7 dorsal root ganglia. The nuclei and nucleoli of the dorsal root ganglia nerve cells were studied by the usual standard transmission electron microscopic procedures. Comparing the control and ischemized material we have observed the different reaction of the nuclei and nucleoli to the ischemia. Some of these structures remained unchanged, the others demonstrated a various degree of the alteration. After the partial ischemia some nucleoli were more compact, some nucleolonemata were less distinct but no karyoplasmic changes were noted. The nucleolar interstitial vacuoles were occasionally enlarged and the satellite bodies were observed scarcely in the proximity of the nucleolus.