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.     

Localization of nitric oxide synthase, NADPH diaphorase and soluble guanylyl cyclase in adult rabbit retina

Nitric oxide (NO) acts as a neuronal messenger which activates soluble guanylyl cyclase (SGC) in neighboring cells and produces a wide range of physiological effects in the central nervous system (CNS). Using immunocytochemical and histochemical stains, we have characterized the NO/SGC system in the rabbit retina and to a lesser extent, in monkey retina. Based on staining patterns observed with an antibody to nitric oxide synthase (NOS) type I and a histochemical marker for NADPH diaphorase, a metabolic intermediate required for NOS activity, three major classes of neurons appear to generate NO in the rabbit retina. These include two subclasses of sparsely distributed wide field amacrine cells, rod and cone photoreceptors, and a subpopulation of ganglion cells. Equivalent cell populations were labeled in monkey retina. An antibody to SGC (tested only in rabbit retina), labeled large arrays of cone photoreceptors in the outer nuclear layer, both amacrine and bipolar cells in the inner nuclear layer (INL), as well as populations of neurons in the ganglion cell layer. These data suggest that the ability to generate NO is restricted to relatively few neurons in the inner retina and to photoreceptor cells in the outer retina; while presumptive target cells, containing pools of SGC, are widespread and form contiguous fields across the inner and outer nuclear layers (ONL) as well as the ganglion cell layer.     

Localization of last-order premotor interneurons in the lumbar spinal cord of rats

There is strong evidence that neural circuits underlying certain rhythmic motor behaviors are located in the spinal cord. Such local central pattern generators are thought to coordinate the activity of motoneurons through specific sets of last-order premotor interneurons that establish monosynaptic contacts with motoneurons. After injections of biotinylated dextran amine into the lateral and medial motor columns as well as the ventrolateral white matter at the level of the upper and lower segments of the lumbar spinal cord, we intended to identify and localize retrogradely labelled spinal interneurons that can likely be regarded as last-order premotor interneurons in rats. Regardless of the location of the injection site, labelled interneurons were revealed in laminae V-VIII along a three- or four-segment-long section of the spinal gray matter. Although most of the stained cells were confined to laminae V-VIII in all cases, the distribution of neurons within the confines of this area varied according to the site of injection. After injections into the lateral motor column at the level of the L4-L5 segments, the labelled neurons were located almost exclusively in laminae V-VII ipsilateral to the injection site, and the perikarya were distributed throughout the entire mediolateral extent of this area. Interneurons projecting to the lateral motor column at the level of the L1-L2 segments were also located in laminae V-VII, but most of them were concentrated in the middle one-third or in the lateral half of this area. Following injections into the medial motor column at the level of the L1-L2 segments, the majority of labelled neurons were confined to the medial aspect of laminae V-VII and lamina VIII, and the proportion of neurons that were found contralateral to the injection site was strikingly higher than in the other experimental groups. The results suggest that the organization of last-order premotor interneurons projecting to motoneurons, which are located at different areas of the lateral and medial motor columns and innervate different muscle groups, may present distinct features in the rat spinal cord.     

Localization of glutamate receptors in dorsal horn of rat spinal cord

Immunocytochemical localization of metabotropic glutamate receptors (mGluRs) and ionotropic glutamate receptors (NMDA-type: NMDAR1 and NMDAR2A-C; AMPA-type: GluR1-4) was performed on sections of rat dorsal horn. Immunoreactivity for mGluR1 alpha was detected in laminae I-III of the dorsal horn, whilst mGluR2/3 immunoreactivity was detected primarily in lamina III. Immunoreactivity for NMDAR1, GluR1, GluR2, GluR2/3, GluR4 and GluR5/6/7 was strongly localized in neuronal elements of laminae I-III. Immunoreactivity for NMDAR2B was localized in laminae I-III. No mGluR5, NMDAR2A and NMDAR2C immunoreactivity was detected. In addition, immunoreactivity for receptors was found to co-localize with immunoreactivity for glutamate in the dorsal horn. The present results indicate that glutamate receptors are differentially localized in neuronal elements of dorsal horn where receptor-neurotransmitter interaction takes place.     

NADPH diaphorase activity and nitric oxide synthase immunoreactivity in lordosis-relevant neurons of the ventromedial hypothalamus

The distribution of the enzymes NADPH diaphorase and nitric oxide synthase in the ventromedial nucleus of the hypothalamus of cycling and ovariectomized/estrogen-treated and control female rats was demonstrated using histochemical and immunocytochemical methods. Serial section analysis of vibratome sections through the entire ventromedial nucleus showed that NADPH diaphorase cellular staining was localized primarily in the ventrolateral subdivision. NADPH diaphorase staining was visible in both neuronal perikarya and processes. Light microscopic immunocytochemistry using affinity-purified polyclonal antibodies to brain nitric oxide synthase revealed a similar pattern of labelling within the ventromedial nucleus and within neurons of the ventrolateral subdivision of the ventromedial nucleus. Control experiments involved omitting the primary antibodies; no labelling was visible under these conditions. Some, but not all, neurons in the ventrolateral subdivision of the ventromedial nucleus contained both NADPH diaphorase and brain nitric oxide synthase as demonstrated by co-localization of these two enzymes in individual cells of this area. That NADPH diaphorase and brain nitric oxide synthase were found in estrogen-binding cells was shown by co-localization of NADPH diaphorase and estrogen receptor and brain nitric oxide synthase and estrogen receptor at the light and ultrastructural levels, respectively. Our studies suggest that brain nitric oxide synthase is present and may be subject to estrogenic influences in lordosis-relevant neurons in the ventrolateral subdivision of the ventromedial nucleus. The hypothalamus is a primary subcortical regulatory center controlling sympathetic function. Therefore, not only is nitric oxide likely to be important for reproductive behavior, but also for the regulation of responses to emotional stress and other autonomic functions.     

NADPH diaphorase activity in mammalian retinas is modulated by the state of visual adaptation

NADPH diaphorase histochemistry is commonly used to identify cells containing nitric oxide synthase (NOS), the enzyme catalyzing the production of nitric oxide from L-arginine. NADPH diaphorase activity and NOS immunostaining was demonstrated in different cells of the vertebrate retina; photoreceptors, horizontal cells, amacrine cells, ganglion cells, and Müller cells. However, the physiological role of nitric oxide (NO) in the retina has yet to be elucidated. In this study, we tested the assumption that NADPH diaphorase activity in the retinas of rabbits and rats depended on the state of visual adaptation. In the rabbit, light adaptation enhanced NADPH diaphorase activity in amacrine cells and practically eliminated it in horizontal cells. Dark adaptation induced the opposite effects; the NADPH diaphorase activity was reduced in amacrine cells and enhanced in horizontal cells. Retinas from eyes that were injected intravitreally with L-glutamate exhibited a pattern of NADPH diaphorase activity that was similar to that seen in dark-adapted retinas. In rats, the NADPH diaphorase activity of amacrine and horizontal cells exhibited adaptation dependency similar to that of the rabbit retina. But, the most pronounced effect of dark adaptation in the rat's retina was an enhancement of NADPH diaphorase activity in Müller cells, especially of the endfoot region. Assuming that NADPH diaphorase activity is a marker for NOS, these findings suggest that NO production in the mammalian retina is modulated by the level of ambient illumination and support the notion that NO plays a physiological role in the retina.     

Value of image-guided needle aspiration cytology in the assessment of thoracolumbar and sacrococcygeal masses

We have used a primary cloning assay to determine the frequency of 6-thioguanine (TG)-resistant tubular epithelial cells in kidney tissue from 72 human donors ranging in age from 2 to 94 years. The frequency of TG-resistant mutants ranged from approximately 5 x 10(-5) for donors in the first decade of life to approximately 2.5 x 10(-4) for donors in the eighth and later decades of life. Two different statistical analyses indicated that this increase in mutant frequency is exponential with age. We also observed a 2-fold higher TG-resistant mutant frequency in nephrectomy kidneys containing a coincident renal carcinoma. DNA sequence analyses revealed HPRT gene mutations in each of 14 TG-resistant mutants from seven unrelated donors. Thirteen of these 14 mutants resulted from independent mutational events. These results suggest that somatic mutations are common in renal--and perhaps in other human--epithelia, and thus could play an important role in the genesis of age-associated disease.     

Sparse distribution of NADPH diaphorase neurons in the hippocampal formation of the inbred mutant strain EL mouse

The number of NADPH diaphorase-positive cells in the CA1/CA2 and CA3 regions of Ammon's horn and the subiculum of the hippocampal formation of EL mice, an inbred mutant strain of the ddY mouse susceptible to convulsive seizures, was fewer than that of ddY mice. These findings suggest that smaller numbers of nitric oxide producing cells in the hippocampal formations of EL mice is related to their susceptibility to convulsive seizures.     

Localization of cyclooxygenase-2 and prostaglandin E2 receptor EP3 in the rat lumbar spinal cord

Cyclooxygenase-2 (COX-2) is now considered to be the major constitutively expressed COX isozyme in the central nervous system. The present immunocytochemical study details localization of COX-2 immunoreactivity in rat spinal cord along with the expression of prostaglandin E2 receptor subtype EP3. Prominent COX-2 staining was observed in the nuclear envelope of neurons throughout the spinal cord, especially in the superficial dorsal horn laminae and motoneurons of lamina IX, as well as in glial cells of the white matter. Expression of EP3 receptor was strictly confined to afferent terminal areas in the superficial dorsal horns.     

NADPH diaphorase histochemistry detects inducible nitric oxide synthetase activity in the thymus of naive and staphylococcal enterotoxin B-stimulated mice

Here we examined the changes in NADPH diaphorase (NADPHd) and inducible nitric oxide synthetase (iNOS) positivity in the medulla of the mouse thymus in response to treatment with the superantigen, Staphylococcal enterotoxin B (SEB). A few NADPHd+ and iNOS+ cells scattered in the medulla were detected in the thymi of naive mice. SEB induced the appearance of a large number of NADPHd+- and iNOS-immunoreactive cells in the thymic medulla. In the thymus of iNOS-deficient mice, a total absence of these NADPHd+ and iNOS+ medullary cells was found both under basal conditions and after SEB stimulation. With the NADPHd reaction, only endothelial staining was detected in the thymi of iNOS-deficient mice. Our data indicate that NADPHd+ cells in the thymic medulla express iNOS and that SEB induces iNOS expression in the mouse thymus.     

Localization of dopamine D2 receptor in rat spinal cord identified with immunocytochemistry and in situ hybridization

In the present study the distribution of dopamine D2 receptors in rat spinal cord was determined by means of immunocytochemistry using an anti-peptide antibody, directed against the putative third intracellular loop of the D2 receptor and in situ hybridization (ISH) using a [35S]UTP labelled anti-sense riboprobe. With the immunocytochemical technique, labelling was confined to neuronal cell bodies and their proximal dendrites. Strongest labelling was present in the parasympathetic area of the sacral cord and in two sexually dimorphic motor nuclei of the lumbosacral cord, the spinal nucleus of the bulbocavernosus and the dorsolateral nucleus. Moderately labelled cells were present in the intermediolateral cell column, the area around the central canal and lamina I of the dorsal horn. Weak labelling was present in the lateral spinal nucleus and laminae VII and VIII of the ventral horn. Except for the two sexually dimorphic motornuclei of the lumbosacral cord labelled motoneurons were not encountered. With the ISH technique radioactive labelling was present in many neurons, indicating that they contained D2 receptor mRNA. The distribution of these neurons was very similar to the distribution obtained with immunocytochemistry, but with ISH additional labelled cells were detected in laminae III and IV of the dorsal horn, which were never labelled with immunocytochemistry. The present study shows that the D2 receptor is expressed in specific areas of the rat spinal cord. This distribution provides anatomical support for the involvement of D2 receptors in modulating nociceptive transmission and autonomic control. Our data further indicate that D2 receptors are not directly involved in modulating motor functions with the exception, possibly, of some sexual motor functions.     

Effects of caudal traction of the spinal cord on evoked spinal cord potentials in the cat

This study attempts clarify the mechanism of neurological deficits in tethered cord syndrome using evoked spinal cord potentials (ESCPs). ESCPs in response to both sciatic nerve (SN-ESCP) and spinal cord stimulation (SC-DESCP) were recorded from the dorsal epidural space. With a fixed degree of caudal traction on the spinal cord in ten cats for 2-4 hours, ESCPs were increased in amplitude in the N1 and N2 deflections of the SC-DESCPs to 158% and 154% at L5 and decreased to 91% and 76% after transient augmentation at L3. On the other hand, the amplitude in the N1 deflection of the SN-ESCPs at L3 and L5 was decreased to 40% and 68%. These findings suggest that not only the force but also the duration of traction influence the degree of the spinal cord dysfunction. When the spinal cords of 17 cats received compression with traction and without traction, the SN-ESCPs of the former became positive earlier than that of the latter. The extent of the recovery in amplitude of both SC-DESCPs and SN-ESCPs propagated over compression site was far limited in the former than in the latter. These results would indicate that the spinal cord subjected to traction is vulnerable to compression.     

Modulation of quinolinic acid-induced depletion of striatal NADPH diaphorase and enkephalinergic neurons by inhibition of nitric oxide synthase

The present study was designed to examine the role of nitric oxide (NO) in quinolinic acid (QUIN)-induced depletion of rat striatal nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase and enkephalinergic neurons. Intrastriatal injection of QUIN produced a dose-dependent decrease in NADPH diaphorase and enkephalin positive cells, with cell loss being evident following the injection of 6 and 18 nmol QUIN, respectively. To evaluate the role of NO in QUIN-induced toxicity, animals were pretreated with the non-specific nitric oxide synthase (NOS) inhibitor, Nomega-nitro-l-arginine (l-NAME) or the selective neuronal NOS inhibitor, 7-nitro indazole (7-NI). l-NAME (2x250 mg/kg, i.p. 8 h apart) maximally inhibited striatal NOS activity by 85%, while 7-NI (50 mg/kg, i.p.) maximally inhibited striatal NOS activity by 60%. Pretreatment with l-NAME or 7-NI potentiated the loss of NADPH diaphorase neurons resulting from intrastriatal injection of low doses of QUIN (18 nmol). Neither NOS inhibitor had any effect on the loss of striatal NADPH diaphorase neurons induced by a higher dose of QUIN (24 nmol). In contrast, 7-NI partially prevented the QUIN (18 and 24 nmol)-induced loss of enkephalinergic neurons, while l-NAME had no effect. These results indicate that NO formation may play a role in QUIN-induced loss of enkephalinergic neurons, but not in the loss of NADPH diaphorase neurons.     

Effect of spinal cord transection on N-methyl-D-aspartate receptors in the cord

Spinal cord injury can lead to an exaggeration of transmission through spinal pathways, resulting in muscle spasticity, chronic pain, and abnormal control of blood pressure and bladder function. These conditions are mediated, in part, by N-methyl-D-aspartate (NMDA) receptors on spinal neurons, but the effects of cord injury on the expression or function of these receptors is unknown. Therefore, antibodies to the NMDA-R1 receptor subunit and binding of [3H]MK-801 were used to assess NMDA receptors in the spinal cord. Receptor density in rats with intact spinal cords was compared to that in rats 1 and 2 weeks after spinal cord transection (SCT) at the mid-thoracic level. At 1 and 2 weeks after SCT, [3H]MK-801 binding was reduced in most laminae in cord segments caudal to the injury, whereas no decrease in amount of R1 subunit immunoreactivity was observed. No significant changes in [3H]MK-801 binding and NMDA-R1 immunoreactivity could be seen rostral to the transection. Since [3H]MK-801 binding requires an open ion channel, the discrepancy between [3H]MK-801 binding and immunocytochemistry may indicate a loss of functional receptors without a consistent change in their total number. Therefore, the exaggerated reflexes that are well established in rats 2 weeks after cord injury must be mediated by a mechanism that withstands attenuation of NMDA receptor function.     

Epidural spinal cord stimulation in the management of spasms in spinal cord injury: a prospective study

Forty-eight spinal cord injury victims were implanted with an epidural spinal cord stimulation system to treat spasms that had not satisfactorily responded to medical therapy. All the patients were at least 6 months after the injury. The protocol included assessment by independent examiners preoperatively and at 3, 6, 12 and 24 months after the implant. Pre- and postoperative data collection included the frequency and severity of the spasms. Combining the frequency and intensity scores into a 'severity' score provided a more accurate clinical picture. No patient observed neurological deterioration following the surgical procedure or the neurostimulation treatment. A statistically significant reduction in the severity of the spasms was observed in the follow-up evaluations, with results that progressively increased in time. It is appears that spinal cord stimulation is an effective and safe alternative in the management of spasms in spinal cord injury victims. Its exact role in relation to intrathecal baclofen infusion and ablative procedures remains to be defined.     

Localization of bradykinin-like immunoreactivity in the rat spinal cord: effects of capsaicin, melittin, dorsal rhizotomy and peripheral axotomy

A putative role for bradykinin has been proposed in the processing of sensory information at the level of the spinal cord. Autoradiographic studies have demonstrated the presence of B2 kinin receptor binding sites in superficial laminae of the dorsal horn and a down-regulation of those receptors in rat models of pain injury. In this study, classical immunocytochemistry and confocal microscopy immunofluorescence were used first to localize bradykinin-like immunoreactivity in all major spinal cord segments of naive rats; second, to assess bradykinin-like immunoreactivity changes that occur in animals subjected to various chemical treatments and surgical lesions. High densities of bradykinin-like immunoreactivity were observed in motoneuron of the ventral horn, deeper laminae and nucleus dorsalis of the dorsal horn. Higher magnification of ventral horn showed strong immunostaining of motoneuron perikaryas and their proximal processes. Two types of bradykinin-like immunoreactivity immunostained cellular bodies were observed in deeper laminae of the dorsal horn. These interneurons, morphologically corresponding to islets and antenna-type cells project dendrites to adjacent laminae. Furthermore, numerous strongly marked dendrites, transversally cut, suggest the presence of projection neurons to higher cervical centres. Following unilateral lumbar dorsal rhizotomy (L1-L6) or peripheral lesion of the sciatic nerve, important increases of bradykinin-like immunoreactivity were found in laminae III and IV of the ipsilateral dorsal horn. In contrast, significant decreases of immunodeposits were observed in both cell bodies and numerous dendrites of motoneuron surrounding neuropil. Specific destructions of sensory afferent fibres with capsaicin or selective activation of kallikreins with melittin caused increases of bradykinin-like immunoreactivity in both the dorsal and ventral horns of the spinal cord. These results which demonstrate the cellular localization of bradykinin-like immunoreactivity in both dorsal and ventral horns of the rat spinal cord, further reveal the plasticity of this non-sensory peptidergic system following various chemical and surgical treatments. Hence, these anatomical findings along with earlier functional and receptor autoradiographic studies reinforce the putative role of bradykinin in sensory function.