The Effect of Long-Term Reduction of Aortic Blood Flow on Spinal Cord Gray Matter in the Rabbit. Histochemical Study of NADPH-Diaphorase

1. The aim of this work was to study the influence of reduced aortic blood flow on NADPH-diaphorase (NADPH-d) staining in the gray matter of L4–S3 spinal cord segments.2. Surgery was performed on the abdominal aorta of the rabbit. Spinal cord ischemia was introduced by infrarenal aortic constriction to 30% from the normal blood flow. Animals were allowed to survive 1 week, 1 month and 3 months after surgery. Neurological outcome was studied in relation to the duration of aortic occlusion. The NADPH-d histochemistry was used for the visualisation of spinal cord sections.3. The most affected area of the spinal cord was pericentral region, and slight changes were seen in the NADPH-d activities of both dorsal and ventral horns. One week after surgery, NADPH-d positive pericentral neurons were almost unchanged in their shape and intensity of staining, the only difference was seen in slightly increased staining of the background around the central canal. One month following surgery neurons exhibited shrinkage or were swollen, NADPH-d staining was less intensive in the pericentral zone and positively stained vessels were present.4. Three months of ischemia influenced the NADPH-d activity: (a) In the pericentral region were seen intensively NADPH-d stained neurons almost normal in shape of their bodies but with shortened processes or without them; (b) NADPH-d staining of neuropil was greatly enhanced mostly around the central canal and in the dorsal commissure; (c) Numerous vessels were present in the pericentral zone and in the location of the ventral horn.5. It can be concluded that the reduction of blood flow in the abdominal aorta makes most changes in the pericentral region of the rabbit spinal cord. Increased NADPH-d staining of neuropil and the presence of positively stained vessels reflect increased NADPH-d/NOS production in the spinal cord gray matter after long-term incomplete aortic occlusion.     

Effect of partial ischemia and axotomy on activities of cholinergic enzymes in lower motor neurons of the dog

Activities of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in the ventral spinal cord, ventral spinal roots and in the central and peripheral stumps of the sciatic nerve transected under conditions of partial ischemia (produced by aortic ligation just below the renal arteries) were compared to those obtained under intact blood supply in time intervals 5, 10, or 15 days after surgery. The significant increase of ChAT activity in the central part of the sciatic nerve following 15 days of partial ischemia correlated with less significant elevation of ChAT in the ventral spinal cord. The changes of AChE activity were not significant during partial ischemia. ChAT in the peripheral stump of the sciatic nerve following 5 days of partial ischemia was preserved by 40% and AChE by 20% more than under normal blood supply. On the contrary, in the next 5 days interval losses of enzymes activity in the degenerating nerve were greater. ChAT was almost totally inactivated whereas 50% of AChE activity was preserved until the end of period examined.     

Involvement of gicerin, a cell adhesion molecule, in development and regeneration of chick sciatic nerve

We have examined the role of gicerin, an immunoglobulin superfamily cell adhesion molecule, in chick sciatic nerves during development and regeneration. Gicerin was expressed in the spinal cord, dorsal root ganglion (DRG) and sciatic nerves in embryos, but declined after hatching. Neurite extensions from explant cultures of the DRG were promoted on gicerin's ligands, which were inhibited by an anti-gicerin antibody. Furthermore, gicerin expression was upregulated in the regenerating sciatic nerves, DRG and dorsal horn of the spinal cord after injury to the sciatic nerve. These results indicate that gicerin might participate in the development and regeneration of sciatic nerves.     

Neuronal nitric oxide synthase in the rabbit spinal cord visualised by histochemical NADPH-diaphorase and immunohistochemical NOS methods

The NADPH-diaphorase (NADPH-d) staining method is widely used in the investigation of both the central and peripheral nervous systems. Neuronal nitric oxide synthase (nNOS) has previously been shown to be responsible for the NADPH-d activity in neurons. However, NADPH-d activity does not always fully represent the enzyme nNOS. We investigated the distribution of NADPH-d activity and nNOS protein in the rabbit spinal cord for all groups of neurons and Rexed's laminae. In most laminae the distribution of NADPH-d activity was identical to nNOS immunoreactivity. Both were present in the dorsal horn and in pericentral areas of the spinal cord, but some differences existed. The superficial part of the dorsal horn (laminae I-III) stained more intensely for NADPH-d than for nNOS. However, the most prominent difference was seen in the lateral part of the dorsal horn--the lateral collateral pathway (LCP). The LCP stained strongly for NADPH-d activity, while nNOS staining was absent. Although there is an excellent correlation between NADPH-d staining and nNOS immunohistochemical staining in the spinal cord in general, the presence of staining differences necessitates the use of immunohistochemistry for some specialized applications.     

Remote electrodermic response to an algogenic stimulus. Pathway and spinal integration

The pathway of a non-segmental sudomotor reflex was studied in rabbits (New Zealand white). By means of thermic stimulation (45 degrees during 30") at the lateral border of the foot, a sudoral response was evoked in a circumscribed area of the pinna. By sequential sections of different nerves and the nervous network around the saphenous and femoral vessels, it was possible to establish the following afferent pathways to the spinal cord: lateral plantar nerve, tibial nerve up to the tuber calcanei, saphenous perivascular network, femoral perivascular network and femoral nerve. The fibres responsible for the podo-auricular sudomotor reflex penetrate into the spinal cord above L4, because the spinal transection at this level does not alter the auricular response. Since the hemisection of the spinal cord at T6 suppresses this reflex in the pinna of the same side, it must be concluded that the spinal pathway is ipsilateral. The efferent pathway abandons the spinal cord beneath segment C6: in fact, the spinal transection at C6 does not alter the auricular response to plantar stimulation. Finally, the sudomotor impulses reach the pinna sweat glands with the auricular vessels.     

The vulnerability of nitrergic neurons to transient spinal cord ischemia: a quantitative immunohistochemical and histochemical study

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.     

The spatial relationship between the musculature and the NADPH-diaphorase activity, 5-HT and FMRFamide immunoreactivities in redia, cercaria and adult Echinoparyphium aconiatum (Digenea)

The spatial relationship between the musculature and the NADPH-diaphorase (NADPH-d) activity, 5-HT and FMRFamide immunoreactivities in redia, cercaria and adult Echinoparyphium aconiatum was studied using scanning electron microscopy (SEM), NADPH-d histochemistry, immunocytochemistry, and confocal scanning laser microscopy (CSLM). TRITC-conjugated phalloidin was used to stain the musculature. Staining for NADPH-d was observed in the central (CNS) and peripheral nervous system (PNS) of all three stages. NADPH-d positive nerves occurred very close to muscle fibres. 5-HT-immunoreactive (5-HT-IR) nerve cells and fibres occurred in the CNS and PNS and close to muscle fibres. FMRFamide-IR nerve fibres were observed in the CNS and PNS of adult worms. This is the first time, the presence of the NADPH-d has been demonstrated in the larval as well as the adult stages of a fluke.     

NADPH-diaphorase localization in the radial nerve cords of the starfish Patiria pectinifera

The presence and localization of NADPH-diaphorase (NADPH-d) in the radial nerve cords of Patiria pectonifera was shown by electron histochemistry. NADPH-d-positive structures were found in ectoneural and hyponeural regions of the radial nerve cord. Ultrastructural localization of NADPH-d was detected in neurons, sensory cells, supporting cells, and in the nerve plexus. The highest enzymatic activity in ectoneural region of the radial nerve cord is due, presumably, to the involvement of NADPH in sensory signal processing.     

Lateral spinal nucleus of the rat: NADPH-diaphorase activity and Fos expression after noxious peripheral stimulation

Results of electrophysiological studies suggest a significant role of the lateral spinal nucleus (LSpN) in the transmission of nociceptive signals. In our study, the presence of Fos immunoreactivity and NADPH-diaphorase positivity was observed in the rat LSpN following noxious peripheral subcutaneous stimulation. Formalin-induced unilateral hindpaw stimulation in the rat caused bilateral NADPH-d reactivity and ipsilateral Fos expression in this nucleus. In the LSpN of the L3–L5 segments of stimulated rats, on average, 4.1 ± 1.2 NADPH-d-positive, NADPH-d(+), 5.1+1.8 Fos-immunoreactive, Fos(+), and 3.0 ± 1.1 double-labeled neurons per 25-μm-thick section were found unilaterally. A close anatomical relationship between NADPH-d(+) processes and Fos(+) cell nuclei in the LSpN was also observed following noxious peripheral stimulation. These neuroanatomical findings support the hypothesis that the LSpN is involved in pain processing and suggest an important role of nitric oxide-mediated signal transduction in this nucleus. Neirofiziologiya/Neurophysiology, Vol. 40, No. 1, pp. 38–42, January–February, 2008.     

NADPH-Diaphorase-Positive Nerve Cells in Heterotopic Spinal Cord Transplants

The method of ectopic transplantation of embryonic CNS rudiments makes it possible to study the mechanisms underlying adaptation of the transplanted embryonic rudiments. The production of nitric oxide by cells is considered as one of such mechanisms. NADPH-diaphorase is an index of the presence of nitric oxide synthase in cells. It was shown that the nerve cells of rat embryonic spinal cord transplants preserved their capacity to express NADPH-diaphorase after transplantation in the sciatic nerve of an adult animal for six months. The dynamics of NADPH-diaphorase-positive neurons of rat embryonic spinal cord developing after transplantation and in situwere studied. In spinal cord neck region, small bipolar NADPH-diaphorase-positive neurons were visualized on day 17 of prenatal development. After transplantation of the embryonic (day 15) spinal cord in the nerve, NADPH-diaphorase-positive neurons were formed later than in situ: within seven days. The results of histochemical studies carried out within six months after the operation suggest a protective role of NADPH-diaphorase in the neurons of allotransplants developing under the conditions of altered microenvironment and insufficient innervation and also suggest that nitric oxide can cause the death of neurons in long surviving transplants.     

NADPH-diaphorase-positive nerve cells in heterotopic spinal cord transplants

The method of ectopic transplantation of embryonic CNS rudiments makes it possible to study the mechanisms underlying adaptation of the transplanted embryonic rudiments. The production of nitric oxide by cells is considered as one of such mechanisms. NADPH-diaphorase is an index of the presence of nitric oxide synthase in cells. It was shown that the nerve cells of rat embryonic spinal cord transplants preserved their capacity to express NADPH-diaphorase after transplantation in the sciatic nerve of an adult animal for six months. The dynamics of NADPH-diaphorase-positive neurons of rat embryonic spinal cord developing after transplantation and in situ were studied. In spinal cord neck region, small bipolar NADPH-diaphorase-positive neurons were visualized on day 17 of prenatal development. After transplantation of the embryonic (day 15) spinal cord in the nerve, NADPH-diaphorase-positive neurons were formed later than in situ: within seven days. The results of histochemical studies carried out within six months after the operation suggest a protective role of NADPH-diaphorase in the neurons of allotransplants developing under the conditions of altered microenvironment and insufficient innervation and also suggest that nitric oxide can cause the death of neurons in long surviving transplants.     

The spinal cord expression of neuronal and inducible nitric oxide synthases and their contribution in the maintenance of neuropathic pain in mice

Background

Nitric oxide generated by neuronal (NOS1), inducible (NOS2) or endothelial (NOS3) nitric oxide synthases contributes to pain processing, but the exact role of NOS1 and NOS2 in the maintenance of chronic peripheral neuropathic pain as well as the possible compensatory changes in their expression in the spinal cord of wild type (WT) and NOS knockout (KO) mice at 21 days after total sciatic nerve ligation remains unknown.

Methodology/Principal Findings

The mechanical and thermal allodynia as well as thermal hyperalgesia induced by sciatic nerve injury was evaluated in WT, NOS1-KO and NOS2-KO mice from 1 to 21 days after surgery. The mRNA and protein levels of NOS1, NOS2 and NOS3 in the spinal cord of WT and KO mice, at 21 days after surgery, were also assessed. Sciatic nerve injury led to a neuropathic syndrome in WT mice, in contrast to the abolished mechanical allodynia and thermal hyperalgesia as well as the decreased or suppressed thermal allodynia observed in NOS1-KO and NOS2-KO animals, respectively. Sciatic nerve injury also increases the spinal cord expression of NOS1 and NOS2 isoforms, but not of NOS3, in WT and NOS1-KO mice respectively. Moreover, the presence of NOS2 is required to increase the spinal cord expression of NOS1 whereas an increased NOS1 expression might avoid the up-regulation of NOS2 in the spinal cord of nerve injured WT mice.

Conclusions/Significance

These data suggest that the increased spinal cord expression of NOS1, regulated by NOS2, might be responsible for the maintenance of chronic peripheral neuropathic pain in mice and propose these enzymes as interesting therapeutic targets for their treatment.     

低温保存许旺细胞对周围神经再生的作用

目的：比较原代培养许旺细胞（Schwann cells,SCs)和冷冻保存的SCs移植对损伤后坐骨神经再生的作用。方法：原代培养和液氮保存的SCs分别移植到桥接缺损坐骨神经的硅胶管内。在移植后不同时间（第6和8周末）,硅胶管远端神经干内注射HRP,逆行追踪背根神经节和脊髓前角的标记神经元数量;测量再生神经纤维的复合动作电位传导速度;电镜观察再生神经纤维的髓鞘形成。结果：原代培养和冷冻保存SCs在移植后不同时间其背根神经节和脊髓前角神经元HRP标记细胞数量、再生神经纤维的复合动作电位传导速度基本一致,再生神经纤维髓鞘的形成未见明显差别。结论：冷冻保存的SCs仍具有促进损伤后周围神经再生的能力。     

Bilateral activation of STAT3 by phosphorylation at the tyrosine-705 (Y705) and serine-727 (S727) positions and its nuclear translocation in primary sensory neurons following unilateral sciatic nerve injury

Unilateral sciatic nerve compression (SNC) or complete sciatic nerve transection (CSNT), both varying degrees of nerve injury, induced activation of STAT3 bilaterally in the dorsal root ganglia (DRG) neurons of lumbar (L4-L5) as well as cervical (C6–C8) spinal cord segments. STAT3 activation was by phosphorylation at the tyrosine-705 (Y705) and serine-727 (S727) positions and was followed by their nuclear translocation. This is the first evidence of STAT3(S727) activation together with the well-known activation of STAT3(Y705) in primary sensory neurons upon peripheral nerve injury. Bilateral activation of STAT3 in DRG neurons of spinal segments anatomically both associated as well as non-associated with the injured nerve indicates diffusion of STAT3 activation inducers along the spinal cord. Increased levels of IL-6 protein in the CSF following nerve injury as well as activation and nuclear translocation of STAT3 in DRG after intrathecal injection of IL-6 shows that this cytokine, released into the subarachnoid space can penetrate the DRG to activate STAT3. Previous results on increased bilateral IL-6 synthesis and the present manifestation of STAT3 activation in remote DRG following unilateral sciatic nerve injury may reflect a systemic reaction of the DRG neurons to nerve injury.     

周围神经损伤后外源性GKNF对神经元的保护作用

采用硅管套接大鼠切断的坐骨神经模型,局部给予胶质细胞源性神经营养因子（ＧＤＮＦ）,应用尼氏染色、酶组织化学染色方法,观察到外源性ＧＤＮＦ能减少脊髓修复侧前角运动神经元死亡的数目,降低脊髓前角运动神经元及脊神经节感觉神经元中胆碱酯酶（ＣＨＥ）及酸性磷酸酶（ＡＣＰ）变化的幅度。这表明外源性ＧＤＮＦ能保护周围神经切断后引起的神经元损伤．     

大鼠坐骨神经切断后外源性bcl-2对脊髓运动神经元的保护作用

采用大鼠坐骨神经切断损伤模型,行神经外膜端端对线缝合,术中依不同组别,动物于神经缝合处远端0．5cm处分别注射人的正义和反义bcl-2重组腺病毒(Ad／s-bcl-2、Ad／as-bcl-2),报道基因重组腺病毒(Ad／lacZ)和生理盐水。术后48h,7d,15d和30d常规灌注固定大鼠,取L4-L6脊髓节段,应用X-gal染色、bel-2原位杂交和免疫组化染色、TUNEL染色以及乙酰胆碱酯酶(AChE)组织化学染色方法,观察到外源基因能在脊髓中表达,同时外源性Ad／s-bcl-2能显著减少L4到L6节段脊髓前角运动神经元凋亡的数目,减少脊髓前角运动神经元中因坐骨神经切断导致的AChE活性的降低幅度,并加快其恢复。而Ad／as-bcl-2可显著增加坐骨神经切断诱导的脊髓前角运动神经元凋亡数目以及AChE活性降低幅度,并延缓其恢复。这些观察结果表明,外源性bcl-2能保护周围神经切断后引起的脊髓运动神经元损伤。     

Comparative analysis of NADPH-diaphorase positive neurons in the rat, rabbit and pheasant thoracic spinal cord. A histochemical study.

The distribution of NADPH-diaphorase (NADPH-d) activity was investigated and compared in the rat, rabbit and pheasant thoracic spinal cord. The investigation of all spinal cord regions (laminae) in three experimental species revealed marked differences in the distribution of NADPH-d activity. Cross sectional analysis of the spinal cord of the rat, rabbit and pheasant confirmed differences in the shape of the gray matter in all examined species. More detailed investigation of Rexed's laminas showed similar distribution of NADPH-d activity in the spinal cord of the rat and rabbit, which were different when compared with the spinal cord of the pheasant. Ventral horn of the rat and rabbit showed no labelling whereas in pheasant this area possessed a number of scattered, intensively stained neurons. In the location of autonomic preganglionic neurons, differences were found as well. In the rat there was seen a number of densely packed, clearly dark blue coloured neurons. Similarly, these neurons were present in the rabbit spinal cord but they were less numerous. No staining was found in this region of pheasant. Pericentral area (lamina X) and intermediate zone (laminaVII) revealed the presence of NADPH-d positive neurons in all examined species although they differed in number and shape of their bodies. The dorsal horn showed the presence of NADPH-d staining in all three animals but its distribution was different in medio-lateral direction. It can be suggested that observed differencies in the presence and distribution of NADPH-d activity across the examined species may reflect different fylogenetic development.     

周围神经损伤后外源性GDNF对神经元的保护作用

采用硅管套接大鼠切断的坐骨神经模型 ,局部给予胶质细胞源性神经营养因子 (GDNF) ,应用尼氏染色、酶组织化学染色方法 ,观察到外源性GDNF能减少脊髓修复侧前角运动神经元死亡的数目 ,降低脊髓前角运动神经元及脊神经节感觉神经元中胆碱酯酶 (CHE)及酸性磷酸酶 (ACP)变化的幅度。这表明外源性GDNF能保护周围神经切断后引起的神经元损伤。     

Botulinum toxin's axonal transport from periphery to the spinal cord

Axonal transport of enzymatically active botulinum toxin A (BTX-A) from periphery to the CNS has been described in facial and trigeminal nerve, leading to cleavage of synaptosomal-associated protein 25 (SNAP-25) in central nuclei. Aim of present study was to examine the existence of axonal transport of peripherally applied BTX-A to spinal cord via sciatic nerve. We employed BTX-A-cleaved SNAP-25 immunohistochemistry of lumbar spinal cord after intramuscular and subcutaneous hind limb injections, and intraneural BTX-A sciatic nerve injections. Truncated SNAP-25 in ipsilateral spinal cord ventral horns and dorsal horns appeared after single peripheral BTX-A administrations, even at low intramuscular dose applied (5 U/kg). Cleaved SNAP-25 appearance in the spinal cord after BTX-A injection into the sciatic nerve was prevented by proximal intrasciatic injection of colchicine (5 mM, 2 μl). Cleaved SNAP-25 in ventral horn, using choline-acetyltransferase (ChAT) double labeling, was localized within cholinergic neurons. These results extend the recent findings on BTX-A retrograde axonal transport in facial and trigeminal nerve. Appearance of truncated SNAP-25 in spinal cord following low-dose peripheral BTX-A suggest that the axonal transport of BTX-A occurs commonly following peripheral application.