Nitric oxide synthase is expressed in experimental malignant glioma and influences tumour blood flow

Summary The distribution and function of nitric oxide synthase (NOS) was studied in the rodent C6 implantation glioma model. Using a histochemical stain for NADPH diaphorase, which colocalises with NOS, morphological studies revealed non homogenous staining of the constituent tumour cells and the neoplastic endothelium. Immunocytochemical staining for macrophages (ED1, ED2) showed dense positivity at the tumour brain interface with more patchy positivity within the tumour mass. This finding suggests that both macrophages, which are known to produce large amounts of NO, and the C6 cells contribute to the NADPH diaphorase positivity. Administration of the NOS inhibitor N^g-nitro-L-argine methyl ester (L-NAME) significantly reduced both tumour (40%) and contralateral local cerebral blood flow (20%) compared to control animals. These findings demonstrate that (i) NOS is present in experimental malignant glioma; (ii) NO mediated mechanisms contribute to tumour blood vessel dilatation and blood flow regulation; and (iii) using this model there is a significant differential sensitivity of the tumour and brain parenchymal vascular beds to a NOS inhibitor. Further investigations are required to determine the potential therapeutic and biological relevance of these findings and the relative contributions of tumour cells, neoplastic endothelium and reactive macrophages to NO mechanisms in gliomas.Presented at the 2nd Meeting of the British Neurosurgical Research Group, Newcastle, March 1995     

The complex role of nitric oxide in the regulation of glomerular ultrafiltration

The complex role of nitric oxide in the regulation of glomerular ultrafiltration. Nitric oxide is an important neurohumoral modulator of glomerular ultrafiltration and renal hemodynamics. Multiple nitric oxide synthase (NOS) isoforms are present within the kidney. However, it is difficult to discern which NOS is most active from prior studies using non-selective NOS blockers. It is recently apparent that NOS activity is important to the activity of tubuloglomerular feedback (TGF) systems, systems that relate tubular reabsorption to the regulation of glomerular ultrafiltration. Neuronal (nNOS) or brain NOS (bNOS; NOS I) is present within the macula densa, the sensing element of TGF systems. Inhibition of NOS activity and specifically bNOS enhances TGF activity. The TGF system also adapts temporally and these events appear to be dependent upon up-regulation of activity of bNOS within the kidney. Temporal adaptation occurs within one to three hours whereby activation of TGF is followed by a gradual return of the glomerular filtration rate (GFR) and renal blood flow toward normal levels. After 24 hours of benzolamide treatment and withdrawal of this agent, glomerular filtration actually increases to supranormal levels and this is prevented by inhibition of bNOS activity. Factors regulating bNOS activity have not been fully clarified. We have recently observed N-methyl-d-aspartate (NMDA) receptors within the kidney that, when inhibited, result in major reductions in renal blood flow and GFR, suggesting an important role for the NMDA receptor in regulation of renal hemodynamics. Future studies will determine whether NMDA receptor is also an important regulator of bNOS activity and the TGF system.     

The morphology of the koniocellular axon pathway in the macaque monkey

The key objective of this study was to determine the distribution and morphology of koniocellular (K) lateral geniculate nucleus (LGN) axons in primary visual cortex (V1) of the macaque monkey. In particular, we were interested in understanding whether subpopulations of K axons exist in this species and, if so, if these subpopulations arise from different K layers of the LGN. Restricted injections of the tracers, biotinilated dextran amine, or Phaseolus vulgaris leucoagglutinin were targeted to specific LGN K layers under electrophysiological guidance and immunocytochemistry was used to visualize labeled axons in cortex that were subsequently reconstructed through serial sections. A total of 36 complete axons and 166 axon segments were reconstructed. Our results identified at least 2 main subpopulations of K axons in macaque V1 based on branching patterns and bouton distribution. Axons that arise primarily from LGN layers K1 and K2 are morphologically simple and tend to branch in cortical layers 1 and 3A. These axons give rise to fewer boutons than seen in axons arising from the dorsal K LGN layers K3-K6. Axons that arise from LGN layers K3-K6 terminate as complex, focused arbors in the cytochrome oxidase (CO) blobs in layer 3Balpha, with only occasional simple projections to the more superficial layers of cortex. Combined with previous observations, our data suggest that there are at least 3 subclasses of K LGN axons in macaque monkey that are similar to K axons identified earlier in both nocturnal simian owl monkeys (Ding and Casagrande 1997) and in prosimian, bush babies (Lachica and Casagrande 1992) suggesting that the LGN K channels that terminate in the CO blobs and in layer 1 are not unique to macaque monkeys but are a common primate feature.     

Degradation of signal timing in cortical areas V1 and V2 of senescent monkeys

Senescence in monkeys results in a degradation of the functional properties of cortical cells as well as prolonged hyperactivity. We have now compared the spontaneous and visually evoked activity levels, as well as the visual response latencies of cells in cortical areas V1 and V2 of young and very old monkeys. We found that V1 cells within layer 4 exhibit normal latencies. In contrast, in other parts of V1 and throughout V2 hyperactivity in old monkeys is accompanied by dramatic delays in both the intracortical and intercortical transfer of information. Extrastriate cortex (area V2) is affected more severely than striate cortex (V1). Delayed information processing in cerebral cortex should contribute to the declines in cortical function that accompany old age.     

Alterations in the nitric oxide synthase binding sites and non-adrenergic, non-cholinergic mediated smooth muscle relaxation in the diabetic rabbit bladder outlet: possible relevance to the pathogenesis of diabetic cystopathy.

PURPOSE: To investigate the effect of diabetes mellitus (DM) on the density and distribution of nitric oxide synthase (NOS) and the smooth muscle responses to non-adrenergic, non-cholinergic (NANC) nerve stimulation and exogenous nitric oxide (NO) in the rabbit lower urinary tract. MATERIALS AND METHODS: Transverse sections of detrusor, bladder neck and urethra, from control and six months alloxan-induced DM New Zealand White rabbits were incubated with a radioligand for NOS ([3H]-L-N(G)-nitroarginine). Densitometric analysis was performed on the autoradiographs. NADPH diaphorase histochemistry was also used as a marker for NOS activity. Responses to NANC nerve stimulation (5 to 20 Hz) and to NO (10(-6) to 3x10(-4) M.) on smooth muscle strips from detrusor, bladder neck and urethra were measured in organ baths. RESULTS: NOS binding sites were significantly (p<0.03) more dense in the bladder neck than in the detrusor in both DM and control groups. In DM bladder neck, NOS binding sites were significantly (p<0.04) increased compared with the controls. NADPH diaphorase activity appeared markedly increased in the detrusor, bladder neck and urethra of DM animals compared with controls. The mean IC50 for exogenous NO in control versus DM were not statistically different in the bladder neck (1.03x10(-4) M versus 9.8x10(-5) M) and urethra (8.1x10(-5) M versus 8.8x10(-5) M), but the relaxations to 5x10(-6) M of NO were significantly impaired (p<0.04) in the DM urethral smooth muscle. NANC nerve-mediated relaxations were significantly impaired (p<0.001) in the DM urethral smooth muscle. CONCLUSIONS: Alterations of both the NOS binding sites and functional responses to NANC nerve stimulation suggest that NO may have a pathophysiological role in the urinary bladder dysfunction associated with DM.     

Expression of nitric oxide synthase in kidney macula densa cells.

The distribution of nitric oxide synthase (NOS), the enzyme by which NO is generated from L-arginine, was investigated in rat kidney. The indirect immunofluorescence technique using a polyclonal antibody against type I NOS was applied, followed by the histochemical NADPH diaphorase staining technique on the same sections in order to demonstrate the enzymatic activity of NOS. Macula densa cells were strongly stained by both techniques, demonstrating abundant NOS in the cytoplasm of these cells. In addition, these findings were confirmed by nonradioactive in situ hybridization, thus demonstrating the corresponding messenger RNA in macula densa cells as well. Our findings provide the morphological basis for a possible role of NO as a mediator substance in signal transfer from distal tubular fluid to glomerular arterioles.     

大鼠阴茎组织中NOS表达及增龄的影响

为探讨大鼠阴茎组织中一氧化氮合酶分布及增龄对其的影响。本文采用ＮＡＤＰＨｄ组化对不同月龄大鼠阴茎组织进行染色。结果显示ＮＯＳ主要分布于大鼠阴茎组织平滑肌细胞,而内皮和社会纤维含量较少;随年龄增加,阴茎组织中ＮＯＳ表达逐渐减弱,各月龄组间差别明显。     

Halothane minimum alveolar anesthetic concentration and neuronal nitric oxide synthase activity of the dorsal horn and the locus ceruleus in rats.

There is some evidence of a relationship between nitric oxide and pain control pathways. However, it is still controversial whether nitric oxide synthase (NOS) inhibitors affect minimum alveolar anesthetic concentration (MAC). We examined the effects of 7-nitro indazole (7-NI), a selective neuronal NOS (nNOS) inhibitor, on halothane MAC. With nicotinamide adenine dinucleotide phosphate diaphorase histochemistry, we also investigated the nNOS activity of the dorsal horn and the locus ceruleus in 26 Sprague-Dawley rats. 7-NI (100, 500, 1000 mg/kg intraperitoneally) reduced halothane MAC to 0.34% +/- 0.12%, 0.1% +/- 0.03%, and 0.05% +/- 0.12%, dose dependently (P < 0.01). 7-NI also reduced the number of nicotinamide adenine dinucleotide phosphate diaphorase-positive cells by 20% to 65% (P < 0.05 or 0.01) and the staining intensity of the axons in the locus ceruleus and lumbar and thoracic spinal cord as compared with the control group. 7-NI reduced the MAC observed with halothane anesthesia, which was accompanied by nNOS activity suppression in the spinal cord and the locus ceruleus. Our results support the hypothesis that the nitric oxide signaling pathway is related to MAC. IMPLICATIONS: We examined the effects of a selective neuronal nitric oxide synthase inhibitor, 7-nitro indazole, on halothane minimum alveolar anesthetic concentration and measured the nitric oxide synthase activity in the spinal cord and the locus ceruleus of Sprague-Dawley rats using nicotinamide adenine dinucleotide phosphate diaphorase staining method. 7-Nitro indazole decreased both the minimum alveolar anesthetic concentration and neuronal nitric oxide synthase activity.     

一氧化氮在肝硬变大鼠低蛋白血症中的作用

目的探讨一氧化氮（ＮＯ）在肝硬变低蛋白血症中的作用。方法利用硫代乙酰胺（ＴＡＡ）腹腔注射大鼠诱导肝功能的变化，并用辅酶Ⅱ（ＮＡＤＰＨ）硫辛酰胺脱氢酶组织化学法显示不同肝功能时期肝脏一氧化氮合成酶（ＮＯＳ）的分布。结果正常鼠肝脏ＮＯＳ染色阴性；大鼠肝功能异常但无低蛋白血症时，肝小叶周边有少量散在的ＮＯＳ阳性细胞，至肝硬变有明显低蛋白血症时，假小叶内肝细胞均为ＮＯＳ染色阳性；停止注射ＴＡＡ４周后，大鼠肝功能逐渐恢复，但仍有低蛋白血症，此时假小叶内肝细胞仍为ＮＯＳ阳性，仅染色稍浅于肝功能明显异常时。结论肝硬变时肝细胞内ＮＯＳ被激活而产生ＮＯ，后者通过抑制肝细胞合成蛋白质而可能参与肝硬变时低蛋白血症的形成。     

Human-specific organization of primary visual cortex: alternating compartments of dense Cat-301 and calbindin immunoreactivity in layer 4A

There is evidence that the cortical anatomy of the magnocellular (M) visual pathway, which carries information about motion and luminance contrast, was modified in human evolution. Recent results indicate that layer 4A of humans contains a meshwork of tissue bands that stain densely for nonphosphorylated neurofilament (NPNF), a protein that is preferentially expressed in elements of the M pathway, whereas apes and monkeys lack a comparable pattern. Here we examined the distribution of staining for Cat-301 -- a monoclonal antibody well established to stain M-related structures preferentially -- in area V1 of humans, apes (chimpanzees, orangutan), Old World monkeys (macaques) and New World monkeys (spider monkeys, squirrel monkeys). Single-staining experiments, using a peroxidase-tetramethylbenzidine (TMB) reaction, revealed alternating zones of dark and light staining for Cat-301 in layer 4A of humans, similar to those observed with NPNF. Double-staining studies in humans revealed that Cat-301-immunoreactive somas and neuropil were localized within the same tissue bands that stained strongly for NPNF and, furthermore, that these bands alternated with irregularly shaped territories that stained very strongly for calbindin. Nonhuman primates, by contrast to humans, displayed weak Cat-301 and calbindin staining in layer 4A. The co-localization of Cat-301 and NPNF in human layer 4A, and the weak staining for these molecules in layer 4A of other primates, suggests that the cortical representation of the M channel was modified in recent human evolution. The calbindin-rich compartments in human layer 4A cannot be related to a particular geniculostriate pathway on neurochemical grounds; they may constitute an interneuronal population that increased in human evolution.     

Nitric oxide synthase is absent in only a subset of cases of pyloric stenosis

PURPOSE: The aim of this study was to study nitric oxide synthase (NOS) immunohistochemistry in the pyloric muscle and establish the role of nitric oxide in pyloric stenosis. METHODS: Pyloric muscle biopsy specimens were obtained from 20 patients with pyloric stenosis during pyloromyotomy. Ten control specimens without pyloric disease were obtained from autopsy performed less than 4 hours after death on age-matched babies who died of other causes. Tissues were fixed in 4% paraformaldehyde immediately. A monoclonal antibody against the neuronal form of NOS (bNOS) was used for immunohistochemistry. RESULTS: Immunohistochemistry showed activity of bNOS in the control specimens and some pyloric stenosis specimens. This shows that NOS is present in the pylorus in normal cases as well as in a few cases of pyloric stenosis. CONCLUSIONS: NOS deficiency leading to lack of locally available nitric oxide causes a failure of smooth muscle relaxation. This may account for the cause of pyloric stenosis in infants. However, this study shows that this is true probably only in a subset of cases. The etiology of pyloric stenosis may still be multifactorial. Further investigations are required regarding the etiology of pyloric stenosis. J Pediatr Surg 36:616-619.     

Localization of nitric oxide synthases during fracture healing.

Previously, we have reported that nitric oxide synthases (NOSs), which generate NO, modulate fracture healing. However, the cellular sources of the NOS isoforms during the course of fracture healing have not been studied systematically. The purpose of this study was to localize the cellular distribution of NOS isoforms (inducible NOS [iNOS], endothelial NOS [eNOS], and neuronal NOS [bNOS]) by in situ hybridization and immunohistology after femoral fractures in rats. The iNOS signal was detected during the initial stages (on day 4 and day 7) of fracture healing in 52 +/- 2% (mean +/- SE, n = 7) of cells within the intramembranous region, along the edge of the periosteal callus. The iNOS signal in callus cells declined to an undetectable level on day 14. eNOS was detected during the middle stages (on day 7 and day 14) of fracture healing in cells lining the blood vessels and also in 49 +/- 3% of cells in the chondral region. The bNOS signal was found to be increased at the later stages (day 14 and day 21) of fracture healing in 51 +/- 3% of cells at the junction between fibrous tissue and cartilage within the fibrochondral region. In summary, the,expression of NOS isoforms during fracture healing was time dependent and cellular distinctive.     

Influence of Body Size on Clinical Outcome in Patients Undergoing Coronary Surgery with or Without Cardiopulmonary Bypass

Aim: Saphenous vein (SV) is the most commonly used conduit in bypass procedures but has a one‐year occlusion rate of 15‐30%. A new ‘no‐touch’ technique where the SV is harvested with a cushion of surrounding tissue with no distension has led to improved early patency rates of 5% at 18‐months. Nitric oxide (NO), synthesised by nitric oxide synthase (NOS) has properties beneficial to graft patency. Our aim was to study the distribution of NOS in SV harvested by this technique and the effect of distension and removal of perivascular tissue on NOS content of SV. Methods: Following ethical committee approval and patients' informed consent, SVs were harvested from ten patients undergoing coronary artery bypass grafting. A segment of vein was harvested by the conventional technique (surrounding tissue stripped and vein distended with saline); another part was stripped but not distended (‘control’) and the remaining parts harvested by the ‘no‐touch’ technique. Samples of each segment were taken and transverse sections prepared for NOS identification using ³[H]L‐N^G nitroarginine (NO Arg) autoradiography and NADPH‐diaphorase histochemistry. NOS isoforms were studied using standard immunohistochemistry. Endothelial cells and nerves were also identified using immunohistochemistry with CD31 and NF200 respecitvely, to confirm sources of NOS. Morphometric analysis of NADPH‐diaphorase staining was carried out to study tissue NOS content. Results: NO Arg binding representing NOS was preserved on the lumen of ‘no‐touch’ vessels whilst that on conventional and control vessels was reduced. NOS was also localised to the medial smooth muscle cells of all vein segments and to the intact adventitia of ‘no‐touch’ segments. This was confirmed by NADPH‐diaphorase staining, which revealed a mean reduction of NOS by 19.5% (p < 0.05, ANOVA) in control segments due to stripping of surrounding tissue alone and a reduction of 35.5% (p < 0.01, AVNOVA) in conventional segments due to stripping and distension, compared to ‘no‐touch’ segments. Adventitial NOS sources in ‘no‐touch’ vessels corresponded to vasa vasorum and paravascular nerves. All three NOS isoforms contributed to the preserved NOS in ‘no‐touch’ vessels. Conclusions: Apart from preserved lumenal NOS, NOS sources are also located in the media and adventitia of SV grafts. These are reduced by both adventitial damage and vein distension during conventional vein harvesting. The ‘no‐touch’ technique avoids these procedures, preserving NOS sources. This may result in improved NO availability in SV harvested by this technique, contributing to the improved patency rates reported.     

The connections of layer 4 subdivisions in the primary visual cortex (V1) of the owl monkey

The primary visual cortex (V1) of primates receives signals from parallel lateral geniculate nucleus (LGN) channels. These signals are utilized by the laminar and compartmental [i.e. cytochrome oxidase (CO) blob and interblob] circuitry of V1 to synthesize new output pathways appropriate for the next steps of analysis. Within this framework, this study had two objectives: (i) to analyze the con- nections between primary input and output layers and compartments of V1; and (ii) to determine differences in connection patterns that might be related to species differences in physiological properties in an effort to link specific pathways to visual functions. In this study we examined the intrinsic interlaminar connections of V1 in the owl monkey, a nocturnal New World monkey, with a special emphasis on the projections from layer 4 to layer 3. Interlaminar connections were labeled via small iontophoretic or pressure injections of tracers [horseradish peroxidase, biocytin, biotinylated dextrine amine (BDA) or cholera toxin subunit B conjugated to colloidal gold particles]. Our most significant finding was that layer 4 (4C of Brodmann) can be divided into three tiers based upon projections to the superficial layers. Specifically, we find that 4alpha (4Calpha), 4beta (4Cbeta) and 4ctr send primary projections to layers 3C (4B), 3Bbeta (4A) and 3Balpha (3B), respectively. Examination of laminar structure with Nissl staining supports a tripartite organization of layer 4. The cortical output layer above layer 3Balpha (3B) (e.g. layer 3A) does not appear to receive any direct connections from layer 4 but receives heavy input from layers 3Balpha (3B) and 3C (4B). Some connectional differences also were observed between the subdivisions of layer 3 and the infragranular layers. No consistent differences in connections were observed that distinguished CO blobs from interblobs or that could be correlated with differences in visual lifestyle (nocturnal versus diurnal) when compared with connectional data in other primates. Re-examination of data from previous studies in squirrel and macaque monkeys suggests that the tripartite organization of layer 4 and the unique projection pattern of layer 4ctr are not restricted to owl monkeys, but are common to a number of primate species.     

Nitric oxide modulates fracture healing.

The role of the messenger molecule nitric oxide has not been evaluated in fracture healing. NO is synthesized by three kinds of nitric oxide synthase (NOS): inducible NOS (iNOS), endothelial (eNOS), and neuronal (bNOS). We evaluated the role of these enzymes in a rat femur fracture-healing model. There was no messenger RNA (mRNA) expression, immunoreactivity, or enzymatic activity for NOS in unfractured femoral cortex. After fracture, however, mRNA, protein, and enzymatic activity for iNOS were identified in the healing rat femoral fracture callus, with maximum activity on day 15. The mRNA expression for eNOS and bNOS was induced slightly later than for iNOS, consistent with a temporal increase in calcium-dependent NOS activity that gradually increased up to day 30. mRNA expression for the three NOS isoforms also was found in six of six human fracture callus samples. To study the effect of suppression of NO synthesis on fracture healing, an experimental group of rats was fed an NOS inhibitor, L-nitroso-arginine methyl ester (L-NAME), and the control group was fed its inactive enantiomer, D-nitroso-arginine methyl ester (D-NAME). An 18% (p < or = 0.01) decrease in cross-sectional area and a 45% (p < or = 0.05) decrease in failure load were observed in the NOS-inhibited group on day 24 after fracture. Furthermore, the effect of NO supplementation to fracture healing was studied by delivering NO to the fracture site using carboxybutyl chitosan NONOate locally. On day 17 after fracture, there was a 30% (p < or = 0.05) increase in cross-sectional area in the NO-donor group compared with the NOS inhibition group. These results show for the first time that NO is expressed during fracture healing in rats and in humans, that suppression of NOS impairs fracture healing, and that supplementation of NO can reverse the inhibition of healing produced by NOS inhibitors.     

Neuronal responses in visual area V2 (V2) of macaque monkeys with strabismic amblyopia

Amblyopia, a developmental disorder of spatial vision, is thought to result from a cascade of cortical deficits over several processing stages beginning at the primary visual cortex (V1). However, beyond V1, little is known about how cortical development limits the visual performance of amblyopic primates. We quantitatively analyzed the monocular and binocular responses of V1 and V2 neurons in a group of strabismic monkeys exhibiting varying depths of amblyopia. Unlike in V1, the relative effectiveness of the affected eye to drive V2 neurons was drastically reduced in the amblyopic monkeys. The spatial resolution and the orientation bias of V2, but not V1, neurons were subnormal for the affected eyes. Binocular suppression was robust in both cortical areas, and the magnitude of suppression in individual monkeys was correlated with the depth of their amblyopia. These results suggest that the reduced functional connections beyond V1 and the subnormal spatial filter properties of V2 neurons might have substantially limited the sensitivity of the amblyopic eyes and that interocular suppression was likely to have played a key role in the observed alterations of V2 responses and the emergence of amblyopia.     

Effect of ischemic preconditioning on hepatic microcirculation and function in a rat model of ischemia reperfusion injury

Ischemic preconditioning (IPC) may protect the liver from ischemia reperfusion injury by nitric oxide formation. This study has investigated the effect of ischemic preconditioning on hepatic microcirculation (HM), and the relationship between nitric oxide metabolism and HM in preconditioning. Rats were allocated to 5 groups: 1. sham laparotomy; 2. 45 minutes lobar ischemia followed by 2-hour reperfusion (IR); 3. IPC with 5 minutes ischemia and 10 minutes reperfusion before IR; 4. L-arginine before IR; and 5. L-NAME + IPC before IR. HM was monitored by laser Doppler flowmeter. Liver transaminases, adenosine triphosphate, nitrites + nitrates, and guanosine 3'5'-cyclic monophosphate (cGMP) were measured. Nitric oxide synthase (NOS) distribution was studied using nicotinamide adeninine dinucleotide phosphate (NADPH) diaphorase histochemistry. At the end of reperfusion phase, in the IR group, flow in the HM recovered partially to 25.8% of baseline (P < .05 versus sham), whereas IPC improved HM to 49.5% of baseline (P < .01 versus IR). With L-arginine treatment, HM was 31.6% of baseline (NS versus IR), showing no attenuation of liver injury. In the preconditioned group treated with L-NAME, HM declined to 10.2% of baseline, suggesting not only a blockade of the preconditioning effect, but also an exacerbated liver injury. Hepatocellular injury was reduced by IPC, and L-arginine and was increased by NO inhibition with L-NAME. IPC also increased nitrate + nitrate (NOx) and cGMP concentrations. NOS detected by NADPH diaphorase staining was associated with hepatocytes and vascular endothelium, and was induced by IPC. IPC induced NOS and attenuated HM impairment and hepatocellular injury. These data strongly suggest a role for nitric oxide in IPC. (Liver Transpl 2002;8:1182-1191.)