Gene and protein expressions of nitric oxide synthases in ischemia-reperfused peripheral nerve of the rat

Wen-Ning Qi2, Zuo-Qin Yan1, Peter G. Whang1, Qi Zhou2, Long-En Chen1, Anthony V. Seaber1, Jonathan S. Stamler3, and James R. Urbaniak1

1 Orthopaedic Microsurgery Research Laboratory, Department of Surgery, 2 Orthopaedic Cell Biology Laboratory, and 3 Howard Hughes Medical Institute, Pulmonary and Cardiovascular Divisions, Department of Medicine, and Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study examined mRNA and protein expressions of neuronal (nNOS), inducible (iNOS), and endothelial nitric oxide synthases (eNOS) in peripheral nerve after ischemia-reperfusion (I/R). Sixty-six rats were divided into the ischemia only and I/R groups. One sciatic nerve of each animal was used as the experimental side and the opposite untreated nerve as the control. mRNA levels in the nerve were quantitatively measured by competitive PCR, and protein was determined by Western blotting and immunohistochemical staining. The results showed that, after ischemia (2 h), both nNOS and eNOS protein expressions decreased. After I/R (2 h of ischemia followed by 3 h of reperfusion), expression of both nNOS and eNOS mRNA and protein decreased further. In contrast, iNOS mRNA significantly increased after ischemia and was further upregulated (14-fold) after I/R, while iNOS protein was not detected. The results reveal the dynamic expression of individual NOS isoforms during the course of I/R injury. An understanding of this modulation on a cellular and molecular level may lead to understanding the mechanisms of I/R injury and to methods of ameliorating peripheral nerve injury.

nitric oxide synthase transcription; nitric oxide synthase translation; reperfusion; sciatic nerve


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NITRIC OXIDE (NO) acts as a neurotransmitter (22), and NO synthase (NOS) isozymes have been shown in the central nervous system (30), dorsal root ganglia (DRG) (7, 37), autonomic nerve fibers (22), and neuromuscular end plates (3, 21). Evidence indicates that NO is also a mediator of nerve injury (12, 41) or the physiological response to injury and may be responsible for abnormal neural activity and pain behavior in animals (39). In the peripheral nervous system, studies have focused on the effects of axonal injury, especially on neurons in the DRG and spinal cord. After ligation of nerve roots, NOS activity was bilaterally decreased in spinal cord and ipsilaterally increased in DRG (7). Peripheral nerve sectioning, however, resulted in an increase in NOS messenger ribonucleic acid (mRNA) in the corresponding DRG (37, 39), which appeared within 2 days and persisted for >2 mo after operation (37).

Alternatively, NO may be involved in regeneration of injured peripheral nerve (44). Data have shown that the three forms of NOS are overexpressed at day 2 after rat sciatic nerve ligature, reaching their greatest expressions during the 2nd wk (14). In particular, long-lasting (4 wk) NOS activity in growth cones and the coexpression of NOS and growth-associated protein (GAP-43) suggest that NO contributes to regeneration (15). However, conflicting results have also been reported. In a mouse study, an NOS inhibitor enhanced regeneration of the proximal stump of transected sciatic nerve, suggesting that NO may have a negative impact on the regeneration (43). NOS mRNA was found in motoneurons of the avulsed root segment where most motoneurons had died. However, this expression was completely inhibited in all motoneurons that regenerated into the peripheral nerve graft (40).

Numerous studies have attempted to characterize the function of NO in ischemia-reperfusion (I/R) injury of the central and peripheral nervous systems; however, no consensus has been reached (17, 30). Hypoxia induced a 10-fold increase of nNOS mRNA expression in nodose ganglia and a 2-fold increase in the cerebellum (32). However, there are no relevant studies specifically addressing the significance of NO in I/R injury of peripheral somatic nerves. We therefore undertook this study to identify the changes in mRNA and protein expressions of constitutive neuronal (nNOS), endothelial (eNOS), and inducible NOS (iNOS) in rat sciatic nerve during ischemia and during early reperfusion.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Both sciatic nerves from 66 male Sprague-Dawley rats weighing 250-300 g were isolated and divided into two groups. In group 1 (ischemia, n = 26), a 1.5-cm segment of one sciatic nerve underwent 2 h of in situ warm ischemia to match the ischemic duration of the I/R group. Complete ischemia was achieved using two 6-0 nylon sutures to ligate the nerve at the proximal and distal ends of the segment. In group 2 (I/R, n = 40), a specially designed device was applied to produce a low-load crush injury, which mimicked an I/R injury (4, 5). In this group, a 5-mm segment of one sciatic nerve in each animal was subjected to a 100-g crush load for 2 h to simulate ischemia, followed by 3 h of reperfusion, after which the nerves were harvested. In each group, the opposite sciatic nerve of each rat was used as a normal control. The surgical procedures were based on those of Chen et al. (4).

mRNA Isolation and cDNA Synthesis

Procedures were based on our earlier studies (34). Poly(A)+ RNA was extracted from the harvested nerves by using Dynabeads oligo(dT)25 (Dynal, Lake Success, NY) according to the manufacturer's instructions. Briefly, the nerve was lysed in lysis/binding buffer that contained 100 mM Tris · HCl, pH 8.0, 500 mM LiCl, 1% lithium dodecyl sulfate (LiDS), 5 mM dithiothreitol, and 10 mM EDTA (Sigma, St. Louis, MO) and sonicated by an ultrasonic processor (Heat Systems, Farmingdale, NY). The lysate was centrifuged to remove debris and was then combined with the Dynabeads oligo(dT)25. Dynabeads were washed twice in a washing buffer containing 10 mM Tris · HCl, pH 8.0, 150 mM LiCl, 0.1% LiDS, and 1 mM EDTA and once with washing buffer without LiDS. Poly(A)+ RNA was eluted from beads in diethylcarbonate-treated water at 67°C, and RNA concentration was measured by using DNA Dip Stik (Invitrogen, San Diego, CA) immediately according to the manufacturer's instructions. Poly(A)+ RNA was reverse transcribed in a PCR machine (model 2400; Perkin-Elmer, Norwalk, CT). A 20-µl reaction mixture contained 2 ng of mRNA, 2.5 µM oligo(dT)16 as a primer, 5 mM MgCl2, 20 units of RNase inhibitor, 1 mM dNTPs, and 50 units of Moloney murine leukemia virus reverse transcriptase (Perkin-Elmer, Branchburg, NJ). First-strand cDNA was synthesized at 25°C for 10 min, 42°C for 15 min, and 99°C for 5 min. In a selected tube, the reverse transcriptase was omitted to control for amplification from contaminating cDNA or genomic DNA.

Quantitative PCR

Quantitation of NOS mRNA levels was performed by a method that involves simultaneous coamplification of both the target cDNA and a reference template (MIMIC) with a single set of primers (35). MIMIC for nNOS, eNOS, iNOS, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were constructed using a PCR MIMIC construction kit (Clontech Laboratories, Palo Alto, CA). Each PCR MIMIC consists of a heterologous DNA fragment with 5'- and 3'-end sequences that was recognized by a pair of gene-specific primers. The sizes of PCR MIMICs were distinct from those of the native targets. The sequence of oligonucleotide primer pairs used for construction of MIMIC and amplification of NOS isoforms and GAPDH mRNA is listed in Table 1. Aliquots were taken from pooled first-strand cDNA from the same group and constituted one sample for PCR. A series of 10-fold dilutions of known concentrations of the MIMIC were added to PCR amplification reactions containing the first-strand cDNA. PCR MIMIC amplification was performed in 100 µl of a solution containing 1.5 mM MgCl2, 0.4 µM primer, PCR DIG labeling mix (200 µM dNTP), 0.002 µM Taq DNA polymerase, and 0.056 µM TaqStart antibody (Clontech Laboratories). After an initial denaturation at 95°C for 65 s, the cycle condition was 15 s at 95°C and 30 s at 60°C for 45 cycles (NOS) and for 35 cycles (GAPDH). Samples were incubated for an additional 8 min at 72°C before completion. Digoxigenin-labeled PCR products were subjected to electrophoresis on agarose gels and transferred to nylon membranes in 10× saline-sodium citrate. Membranes were air-dried, and chemiluminescent detection was carried out using a Wash and Block Buffer Kit and disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan}-4-yl) phenyl phosphate (Boehringer-Mannheim, Indianapolis, IN). Kodak Biomax MR film was exposed to detect chemiluminescent signals. Autoradiogram band intensity was quantitated by densitometry using NIH Image software. Levels of mRNA were calculated from the point of equal density of the sample and MIMIC PCR products (Fig. 1). NOS mRNA levels were normalized with the levels of GAPDH mRNA present in each sample, which served to control for variations in RNA purification and cDNA synthesis. The relative mRNA expression of NOS in each group was compared with those from the respective normal group and reported as a percentage of normal.

                              
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Table 1.   Oligonucleotides of primers for PCR



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Fig. 1.   Analysis of target mRNA levels by competitive PCR. Top: neuronal nitric oxide synthase (nNOS) of digoxigenin-labeled competitive PCR autoradiogram. Bottom: quantitative analysis of the competitive PCR experiment shown at top. Ordinate, ratio of the target to MIMIC products as determined by band densitometry; abscissa, inverse amount of MIMIC added to the amplification. When the resultant target and MIMIC densitometric values are equal (y = 1), extrapolation can be performed to yield the target amount.

Western Blot

Sciatic nerve was homogenized in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.02% sodium azide, 1 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin) and centrifuged at 100,000 g at 4°C for 1 h. Protein concentration in the supernatant was measured with a bicinchoninic acid kit (Pierce, Rockford, IL), and 50 µg of total protein were loaded onto an 8% SDS-polyacrylamide gel and then transferred onto nitrocellulose membrane (Micron Separations, Westborough, MA). The membrane was blocked with 5% nonfat dry milk in TBS-T (pH 7.6, 20 mM Tris base, 137 mM NaCl, and 0.1% Tween 20) at room temperature for 1 h and then incubated with monoclonal primary antibody for nNOS (1:2,000), eNOS (1:1,000), or iNOS [1:2,500; Transduction Laboratories, Lexington, KY; iNOS monoclonal antibody (MAb) was also obtained from Santa Cruz Biotechnology, Santa Cruz, CA] at 4°C overnight. After three washes in TBS-T buffer, the blot was incubated with 1:4,000 horseradish peroxidase-labeled goat anti-mouse IgG (Calbiochem, La Jolla, CA) for 1 h at room temperature. After washes in TBS-T buffer, the blot was detected with an enhanced chemiluminescence detection kit (Amersham). Kodak film was used to detect chemiluminescent signals.

Immunohistochemistry

Five-micrometer longitudinal and cross sections of the nerve were air-dried at room temperature for 30 min, fixed in 4% paraformaldehyde for 10 min, and then blocked with 0.3% H2O2 in methanol at room temperature for 30 min to quench the endogenous peroxidase activity. The sections were washed for 2 min three times in PBS, incubated with 5% horse serum for 20 min to block nonspecific binding, and then incubated with mouse anti-nNOS (1:800), anti-eNOS (1:800), and anti-iNOS (1:50) monoclonal primary antibody at 4°C overnight. After they were thoroughly washed in PBS, the sections were incubated with biotinylated horse anti-mouse rat absorbed secondary antibody (1:200; Vector Laboratories, Burlingame, CA) at room temperature for 30 min and then detected with Vectastain ABC kit (Vector Laboratories). The sections were then developed with diaminobenzidene (Calbiochem) and counterstained with hematoxylin (Sigma). Mouse isotope-matched IgG was used to replace the primary antibody as a negative control.

Schwann cells were identified with a rabbit anti-S-100 polyclonal antibody (1:1,200; Dako). The immunostaining procedure was the same as that described above, except the blocking buffer was 5% goat serum and the secondary antibody (1:200; Dako) was biotinylated. Rabbit isotope-matched IgG was used as the negative control.

Statistical Comparison

Values are means ± SD of three experiments. Statistical comparisons utilized one-way analysis of variance, with P < 0.05 indicating significance.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Expressions of mRNA and Protein of Three NOS Isoforms in Normal Nerve

mRNA expression. With the use of specific oligonucleotide primers for nNOS, iNOS, and eNOS, the RT-PCR amplified products showed single clear bands of the predicted sizes (Fig. 2). With the use of restriction enzyme digestion, each of the primers gave fragments of expected sizes based on the reported sequences. iNOS mRNA was expressed at a very low level.


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Fig. 2.   RT-PCR results show mRNA expression of 3 NOS isozymes in normal rat sciatic nerve. M, molecular marker; N, nNOS; I, inducible NOS (iNOS); E, endothelial NOS (eNOS).

Protein expression. In the nerve homogenate, a 155-kDa protein band identified by the nNOS MAb comigrated with the nNOS positive control from rat brain, and a 140-kDa protein band identified by an eNOS MAb comigrated with the eNOS positive control from human endothelial cells (Fig. 3). An iNOS MAb purchased from Transduction Laboratories detected a protein band with an approximate size of 160 kDa (Fig. 3); however, this was larger than that of the iNOS positive control from macrophage (130 kDa) and could not be confirmed with an iNOS MAb from Santa Cruz Biotechnology. The absence of a cross-reaction between nNOS MAb and eNOS positive control, between eNOS MAb and nNOS positive control, and between iNOS MAb and eNOS or nNOS positive control suggested that each NOS MAb was isoform specific.


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Fig. 3.   Western blotting results display proteins of nNOS and eNOS isozymes in normal rat sciatic nerve. N, nerve homogenate; B, brain lysate (rat brain pituitary) as a positive control for nNOS; E, human endothelial cell lysate as a positive control for eNOS; M, macrophage cell lysate as a positive control for iNOS.

Immunohistochemistry. Reaction of nerve sections with nNOS MAb revealed a robust brown staining of Schwann cells (myelinated and unmyelinated) that appeared to be cytoplasmic. The identity of Schwann cells was verified by anti-S-100 antibody staining. Most axons also stained, although the staining was less intense than that observed in Schwann cells. The myelin sheath, extracellular matrix, and blood vessels did not appear to stain with nNOS MAb (Fig. 4A). Only the endothelial cells of the microvessels were markedly stained by the eNOS MAb (Fig. 4B), and there was no staining of the axons or Schwann cells. Although iNOS MAb obtained from Transduction Laboratories stained the endothelial cells and the periphery of the Schwann cells (Fig. 4C), this positive staining was not confirmed by the iNOS MAb from Santa Cruz Biotechnology (Fig. 4D). Negative controls using mouse IgG or rabbit IgG, instead of primary antibody, revealed no nonspecific staining.


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Fig. 4.   Immunohistochemical stains for nNOS, iNOS, and eNOS isozymes in normal rat sciatic nerve. A: cross section stained with nNOS monoclonal antibody (MAb). Schwann cells (arrow) were labeled with strong brown color; most axons (arrowhead) were also stained, but not as strongly as those of Schwann cells. B: cross section stained with eNOS MAb. Endothelial cells of microvessels (arrowhead) were labeled intensely. C: cross section stained with one iNOS MAb. Positive staining was seen in endothelial cells (arrowhead) and in the margin of Schwann cells (arrow). D: cross section stained with another iNOS MAb showed negative staining in a normal rat sciatic nerve sample. Magnification ×680. Scale bar, 15 µm.

mRNA Expression of Three NOS Isoforms in Ischemic and Reperfused Nerve

At the end of 2 h of ischemia, nNOS and eNOS mRNA expressions remained at normal levels, but iNOS showed a sixfold (604 ± 150%) increase from normal sciatic nerve of the opposite limb (P < 0.001). After 2 h of ischemia and 3 h of reperfusion, nNOS and eNOS mRNA expressions diminished to 91 ± 30 and 62 ± 25% of normal, respectively. In contrast, iNOS mRNA further increased to 1,382 ± 204% of normal, a 14-fold upregulation. Compared with normal and ischemia-only groups, downregulation of eNOS mRNA expression and upregulation of iNOS mRNA expression were significant (Fig. 5).


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Fig. 5.   Results of quantitative RT-PCR analysis for nNOS, iNOS, and eNOS mRNA from the sciatic nerve of the rat after 2 h of ischemia and 2 h of ischemia followed by 3 h of reperfusion. Values are means ± SD. **P < 0.01, ***P < 0.001 compared with normal. ##P < 0.01, ###P < 0.001 compared with ischemia.

Protein Expression of Three NOS Isoforms in Ischemic and Reperfused Nerve

After 2 h of ischemia, the nNOS protein level determined by Western blots decreased slightly from the level of its normal control from the opposite limb. After 2 h of ischemia and 3 h of reperfusion, nNOS protein expression decreased to 63.9 ± 5.6% of normal, a significant difference from normal (P < 0.001) and the ischemia-only group (P < 0.05). eNOS protein expression decreased in response to both ischemia (80.9 ± 5.1%) and I/R (81.0 ± 4.2%). These changes were significant compared with their respective normal control (P < 0.01; Fig. 6). However, there was no band detected in any group to comigrate with iNOS positive control generated from macrophage.


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Fig. 6.   Results of Western blot analysis for nNOS and eNOS mRNA from the sciatic nerve of the rat after 2 h of ischemia and 2 h of ischemia followed by 3 h of reperfusion. A: summary of Western blot analysis. Values are means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control; #P < 0.05 compared with ischemia. B: representative immunoblotting bands of nNOS with anti-nNOS antibody and eNOS with anti-eNOS antibody. nNOS is expressed at 155 kDa and eNOS protein at 140 kDa. PC, positive control; I, ischemic nerve; IR, reperfused nerve; CN, control nerve.

The immunohistochemical staining showed nNOS and eNOS in sections of the ischemia and I/R groups and no significant differences in the patterns of staining among the ischemia-only, I/R, and normal control groups. iNOS staining was not observed in any group.


    DISCUSSION
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Using quantitative RT-PCR and Western blotting, we have demonstrated that ischemia and I/R differentially regulate the expressions of NOS mRNA and protein. After 2 h of ischemia, nNOS and eNOS protein expressions decreased. After 3 h of reperfusion, expression of nNOS and eNOS mRNA and protein decreased further. In contrast, iNOS mRNA significantly increased after ischemia and was further upregulated after I/R, while iNOS protein was not detected by either Western blotting or immunohistochemical staining.

No studies have examined altered expressions of NOS genes and proteins in the somatic nerve after I/R, although evidence suggests an important role for NOS in peripheral nerve injury. Our results support the hypothesis that NO may be involved in the mechanism of tissue injury including I/R injury (6). We have previously shown that supplementation of a NO donor significantly improves the microcirculation and contractile function of reperfused skeletal muscle (6, 25), whereas a NOS inhibitor exaggerates contractile dysfunction (18). That I/R induced significant reduction in the expression of eNOS mRNA and protein in the present study extends the findings of our earlier studies in skeletal muscle in which I/R downregulated eNOS mRNA to 28% of normal (34) and provides rationale for salvage NO therapy. The decrease in eNOS may play a role in exaggerating vessel spasm, neutrophil adhesion, and functional deficits in the early stages of reperfusion, thereby resulting in tissue injury and reperfusion failure. eNOS is mainly produced by endothelial cells and may be produced by Schwann cells (24, 27). Decreased eNOS mRNA and protein expressions may be linked to reperfusion-induced downregulation and/or reperfusion-induced cell damage with resultant failure to express eNOS mRNA.

The regulation of nNOS is exceedingly complex (38). Axotomy results in upregulation of nNOS mRNA in injured hypoglossal motoneurons (28) and increased nNOS immunoreactivity in pudendal motor neurons (33). However, the expression of nNOS mRNA in the present study was slightly downregulated after 2 h of ischemia and 3 h of reperfusion, and protein expression decreased after ischemia and even more after I/R. Evidently, the response to ischemia and I/R differs from that of hypoxia, which increased nNOS mRNA expression in nodose ganglion (32), although with the caveat that we only followed nNOS levels for 5 h. Although nNOS has been implicated in a wide variety of pathophysiological processes, the biological relevance of its decrease in I/R injury of the somatic nerve is unclear at this time, and further study is warranted.

Our data identify iNOS mRNA in normal somatic nerve of the rat, although expression is very low. The results extend our earlier findings in skeletal muscle (34) and, more generally, concur with results showing that the iNOS gene is expressed constitutively in airway epithelium, retina, cerebellum, and skeletal muscle tissue (4, 13, 28, 31, 33). iNOS is typically induced by cytokines and endotoxins and is thought to represent a physiological response of the immune system to trauma, infection, or tissue damage (31). Studies have shown that transcription and translation levels of iNOS were raised in rat sciatic nerve after a chronic constriction injury (23). Thus it is not surprising that iNOS mRNA was increased after ischemia and I/R in this study. iNOS is derived from macrophage, neutrophil, and Schwann cells. Thus we propose that iNOS mRNA during I/R may derive from both resident and infiltrating neutrophil and macrophage cells (19), as well as nerve cells (26). Two cytokines, tumor necrosis factor and interleukin-1, have been shown to be present in injured nerve (8, 29) and may be the source of induction.

Although iNOS mRNA was upregulated 14-fold from normal after 2 h of ischemia and 3 h of reperfusion, it was surprising to us that iNOS protein was not identified in either normal or treated nerve by Western blotting and immunohistochemistry. Several possible explanations should be considered. First, a time delay may be present between an increase in the rate of transcription and a concomitant change in the level of translation. Thus a corresponding increase in iNOS protein levels may occur on a longer time scale than our study. Second, posttranscriptional regulatory mechanisms to decrease protein expression or increase protein degradation may exist (1, 20). Third, it is conceivable that Western blotting and immunohistochemistry are simply not sensitive enough to detect iNOS protein in normal nerve or in tissue subjected to these experimental conditions. Nonetheless, we have little evidence for iNOS expression at the level of protein, suggesting a lesser role in either normal physiology or pathophysiology of early reperfusion of peripheral nerve.

Compared with our earlier results in skeletal muscle, ischemia and I/R resulted in an increased expression of iNOS mRNA, but the extent was much higher in the nerve (up to 14-fold) than in skeletal muscle (only 2.4-fold). The divergent results may be due to differences in the type of target tissue. A similar discrepancy exists in the literature: the effects of hypoxia on gene expression were not uniform among different cells and/or tissues (32, 36, 42).

In the early stage of NO research, it was assumed that the protective vs. destructive role of NO depended on concentration and isoform; that is, high NO concentrations produced by iNOS were implicated in damage to host cells (2, 9-11, 16, 26). Increasing evidence, however, suggests that NO may be beneficial or detrimental depending on the stage of evolution of the event (such as ischemia and I/R) and on the cellular compartment producing NO (11, 16), and injury (e.g., apoptotic mechanism) can be independent of NOS isoform or NO concentration. Our findings of the differential expression of distinct NOS mRNA and protein during ischemia and during early reperfusion in this study support this concept. Furthermore, the results of decreased eNOS mRNA and protein and increased iNOS mRNA in the I/R group in this study suggest the importance of determining the cellular and molecular mechanisms of transcription expressions of individual NOS.

In addition to three NOS mRNAs present in normal somatic nerve, we find nNOS protein in Schwann cells and axons. nNOS is thought to function as a neurotransmitter (2, 30) and may also be involved in the response to nerve injury (12, 38, 41) and regeneration (14, 15, 43, 44). It is difficult to understand, however, how NO, a short-lived molecule that is produced in the neuron cell body, can reach the distal axon. One alternative is that NO is locally synthesized. Indeed, our findings of nNOS in Schwann cells and axons appear to support this possibility. Schwann cells have an intimate relationship with axons and are involved in nutrition, impulse conduction, and nerve regeneration (11), perhaps by producing NO. The findings of nNOS-positive myelinated fibers (motor fibers) in this study contrast, however, with the data from normal spinal cord, where motor neurons are apparently nNOS negative (10). The precise mechanism of NO production and the exact role of NO in the function of somatic nerves, however, remain unknown.

In conclusion, the present study has demonstrated that three NOS genes (nNOS, iNOS, and eNOS) and nNOS and eNOS proteins are expressed in normal somatic nerve and are regulated by ischemia and I/R. The finding that I/R downregulates constitutive expression of NOS, especially eNOS, suggests a potential clinical benefit of NO donor in I/R injury.

Perspectives

Using a rat sciatic nerve I/R model in which the expression of mRNA and protein of three NOS isoforms was determined, we found that ischemia and reperfusion downregulated the expression of both mRNA and protein of nNOS and eNOS but upregulated iNOS mRNA. From a clinical perspective, future study should be directed toward determining dynamic expression of NOS isoforms; in addition, the microdomains in which individual NOS isoforms operate in I/R injury need to be more precisely characterized. An understanding of this modulation on a cellular and molecular level may lead to understanding the mechanisms of the reperfusion injury observed in vivo and to methods of ameliorating clinical outcome of peripheral nerve injury.


    ACKNOWLEDGEMENTS

The authors thank Laura P. Hale and Jie Li (Dept. of Pathology, Duke University Medical Center) for consulting and technical assistance with immunohistochemical evaluation.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-36046 (to J. R. Urbaniak) and HL-59130 (to J. S. Stamler).

Address for reprint requests and other correspondence: L.-E. Chen, Orthopaedic Microsurgery Laboratory, Box 3093, Duke University Medical Center, Durham, NC 27710 (E-mail: chen0006{at}mc.duke.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6 February 2001; accepted in final form 11 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Amin, AR, Patel RN, Thakker GD, Lowenstein CJ, Attur MG, and Abramson SB. Post-transcriptional regulation of inducible nitric oxide synthase mRNA in murine macrophages by doxycycline and chemically modified tetracyclines. FEBS Lett 410: 259-264, 1997[ISI][Medline].

2.   Bredt, DS, and Snyder SH. Nitric oxide, a novel neuronal messenger. Neuron 8: 3-11, 1992[ISI][Medline].

3.   Chao, DS, Silvagno F, Xia H, Cornwell TL, Lincoln TM, and Bredt DS. Nitric oxide synthase and cyclic GMP-dependent protein kinase concentrated at the neuromuscular endplate. Neuroscience 76: 665-672, 1997[ISI][Medline].

4.   Chen, LE, Seaber AV, Glisson RR, Davies H, Murrell GAC, Anthony DC, and Urbaniak JR. The functional recovery of peripheral nerve following defined acute crush injuries. J Orthop Res 10: 657-664, 1992[ISI][Medline].

5.   Chen, LE, Seaber AV, Glisson RR, and Urbaniak JR. The influence of magnitude and duration of crushing load on functional recovery of the peripheral nerve. J Reconstr Microsurg 9: 299-306, 1993[ISI][Medline].

6.   Chen, LE, Seaber AV, Nasser RM, Stamler JS, and Urbaniak JR. The effects of the NO donor S-nitroso-N-acetylcysteine on contractile function of normal and ischemia/reperfused skeletal muscle. Am J Physiol Regulatory Integrative Comp Physiol 274: R822-R829, 1998[Abstract/Free Full Text].

7.   Choi, Y, Raja SN, Moore LC, and Tobin JR. Neuropathic pain in rats is associated with altered nitric oxide synthase activity in neural tissue. J Neurol Sci 138: 14-20, 1996[ISI][Medline].

8.   Creange, A, Barlovatz-Meimon G, and Gherardi RK. Cytokines and peripheral nerve disorders. Eur Cytokine Netw 8: 145-151, 1997[ISI][Medline].

9.   Dawson, VL, and Dawson TM. Nitric oxide neurotoxicity. J Chem Neuroanat 10: 179-190, 1996[ISI][Medline].

10.   Dun, NJ, Dun SL, Wu SY, Forstermann U, Schmidt HHHW, and Tseng LF. Nitric oxide synthase immunoreactivity in the rat, mouse, cat and squirrel monkey spinal cord. Neuroscience 54: 845-857, 1993[ISI][Medline].

11.   Dyck, PJ, and Thomas PK. Peripheral Neuropathy (3rd ed.). Philadelphia, PA: Saunders, 1993, p. 317-330.

12.   Fiallos-Estrada, CE, Kummer W, Mayer B, Bravo R, Zimmermann M, and Herdegen T. Long-lasting increase of nitric oxide synthase immunoreactivity, NADPH-diaphorase reaction and c-JUN co-expression in rat dorsal root ganglion neurons following sciatic nerve transection. Neurosci Lett 150: 169-173, 1993[ISI][Medline].

13.   Gath, I, Closs EI, Godtel-Armbrust U, Schmitt S, Nakane M, Wessler I, and Gorstermann U. Inducible NO synthase II and neuronal NO synthase I are constitutively expressed in different structures of guinea pig skeletal muscle: implications for contractile function. FASEB J 10: 1614-1620, 1996[Abstract/Free Full Text].

14.   Gonzalez-Hernandez, T, and Rustioni A. Expression of three forms of nitric oxide synthase in peripheral nerve regeneration. J Neurosci Res 55: 198-207, 1999[ISI][Medline].

15.   Gonzalez-Hernandez, T, and Rustioni A. Nitric oxide synthase and growth-associated protein are coexpressed in primary sensory neurons after peripheral injury. J Comp Neurol 404: 64-74, 1999[ISI][Medline].

16.   Iadecola, C. Bright and dark sides of nitric oxide in ischemic brain injury. Trends Neurosci 20: 132-139, 1997[ISI][Medline].

17.   Iadecola, C, Zhang F, Xu S, Casey R, and Ross ME. Inducible nitric oxide synthase gene expression in brain following cerebral ischemia. J Cereb Blood Flow Metab 15: 378-384, 1995[ISI][Medline].

18.   Joneschild, ES, Chen LE, Seaber AV, and Urbaniak JR. The effect of a NOS inhibitor, L-NMMA, on contractile functions of reperfused skeletal muscle. J Reconstr Microsurg 15: 55-60, 1999[ISI][Medline].

19.   Keller, AM, Clancy RM, Barr ML, Marboe CC, and Cannon PJ. Acute reoxygenation injury in the isolated rat heart: role of resident cardiac mast cells. Circ Res 63: 1044-1052, 1988[Abstract].

20.   Kunz, D, Walker G, Eberhardt W, and Pfeilschifter J. Molecular mechanisms of dexamethasone inhibition of nitric oxide synthase expression in interleukin-1beta -stimulated mesangial cells: evidence for the involvement of transcriptional and posttranscriptional regulation. Proc Natl Acad Sci USA 93: 255-259, 1996[Abstract/Free Full Text].

21.   Kusner, LL, and Kaminski HJ. Nitric oxide synthase is concentrated at the skeletal muscle endplate. Brain Res 730: 238-242, 1996[ISI][Medline].

22.   Lefebvre, RA. Nitric oxide in the peripheral nervous system. Ann Med 27: 379-388, 1995[ISI][Medline].

23.   Levy, D, Hoke A, and Zochodne DW. Local repression of inducible nitric oxide synthase in an animal model of neuropathic pain. Neurosci Lett 260: 207-209, 1999[ISI][Medline].

24.   Levy, D, and Zochodne DW. Local nitric oxide synthase activity in a model of neuropathic pain. Eur J Neurosci 10: 1846-1855, 1998[ISI][Medline].

25.   Liu, K, Chen LE, Seaber AV, and Urbaniak JR. S-nitroso-N-acetylcysteine protects skeletal muscle against reperfusion injury. Microsurgery 18: 299-305, 1998[ISI][Medline].

26.   Lowenstein, CJ, Dinerman JL, and Snyder SH. Nitric oxide: a physiologic messenger. Ann Intern Med 120: 227-237, 1994[Abstract/Free Full Text].

27.   Ma, L, Morita I, and Murota S. Presence of constitutive type nitric oxide synthase in cultured astrocytes isolated from rat cerebra. Neurosci Lett 174: 123-126, 1994[ISI][Medline].

28.   Nakagomi, S, Kiryu-Seo S, Kimoto M, Emson PC, and Kiyama H. Dimethylarginine dimethylaminohydrolase (DDAH) as a nerve-injury-associated molecule: mRNA localization in the rat brain and its coincident up-regulation with neuronal NO synthase (nNOS) in axotomized motoneurons. Eur J Neurosci 11: 2160-2166, 1999[ISI][Medline].

29.   Oka, N, Akiguchi I, Kawasaki T, Mizutani K, Satoi H, and Kimura J. Tumor necrosis factor-alpha in peripheral nerve lesions. Acta Neuropathol (Berl) 95: 57-62, 1998[ISI][Medline].

30.   Paakkari, I, and Lindsberg P. Nitric oxide in the central nervous system. Ann Med 27: 369-377, 1995[ISI][Medline].

31.   Park, CS, Park R, and Krishna G. Constitutive expression and structural diversity of inducible isoform of nitric oxide synthase in human tissues. Life Sci 59: 219-225, 1996[ISI][Medline].

32.   Prabhakar, NR, Pieramici SF, Premkumar DRD, Kumar GK, and Kalaria RN. Activation of nitric oxide synthase gene expression by hypoxia in central and peripheral neurons. Mol Brain Res 43: 341-346, 1996[ISI][Medline].

33.   Pullen, AN, and Humphreys P. Protracted elevation of neuronal nitric oxide synthase immunoreactivity in axotomized adult pudendal motor neurons. J Anat 194: 547-565, 1999[ISI][Medline].

34.   Qi, WN, Chen LE, Seaber AV, and Urbaniak JR. Regulation of NOS mRNA expression in reperfused muscle. Microsurgery 19: 18-19, 1999.

35.  Qi WN and Scully SP. Extracellular collagen regulates expression of TGF-beta 1 gene. J Orthop Res. In press.

36.   Rydh-Rinder, M, Holmberg K, Elfvin LG, Wiesenfeld-Hallin Z, and Hokfelt T. Effects of peripheral axotomy on neuropeptides and nitric oxide synthase in dorsal root ganglia and spinal cord of the guinea pig: an immunohistochemical study. Brain Res 707: 180-188, 1996[ISI][Medline].

36a.   Singh, I, Grams M, Wang WH, Yang T, Killen P, Smart A, Schnermann J, and Briggs JP. Coordinate regulation of renal expression of nitric oxide synthase, renin, and angiotensinogen mRNA by dietary salt. Am J Physiol Renal Fluid Electrolyte Physiol 270: F1027-F1037, 1996[Abstract/Free Full Text].

37.   Verge, VMK, Xu Z, Xu XJ, Wiesenfeld-Hallin Z, and Hokfelt T. Marked increase in nitric oxide synthase mRNA in rat dorsal root ganglion after peripheral axotomy: in situ hybridization and functional studies. Proc Natl Acad Sci USA 89: 11617-11621, 1992[Abstract].

38.   Wang, Y, Newton DC, and Marsden PA. Neuronal NOS: gene structure, mRNA diversity, and functional relevance. Crit Rev Neurobiol 13: 21-43, 1999[ISI][Medline].

39.   Wiesenfeld-Hallin, Z, Hao JX, Xu XJ, and Hökfelt T. Nitric oxide mediates ongoing discharges in dorsal root ganglion cells after peripheral nerve injury. J Neurophysiol 70: 2350-2353, 1993[Abstract/Free Full Text].

40.   Wu, W, Han K, Li L, and Schinco FP. Implantation of PNS graft inhibits the induction of neuronal nitric oxide synthase and enhances the survival of spinal motoneurons following root avulsion. Exp Neurol 129: 335-339, 1994[ISI][Medline].

41.   Yu, WHA Nitric oxide synthase in motor neurons after axotomy. J Histochem Cytochem 42: 451-457, 1994[Abstract/Free Full Text].

42.   Zhang, X, Verge V, Wiesenfeld-Hallin Z, Ju G, Bredt D, Synder SH, and Hokfelt T. Nitric oxide synthase-like immunoreactivity in lumbar dorsal root ganglia and spinal cord of rat and monkey and effect of peripheral axotomy. J Comp Neurol 335: 563-575, 1993[ISI][Medline].

43.   Zochodne, DW, Levy D, Zwiers H, Sun H, Rubin I, Cheng C, and Lauritzen M. Evidence for nitric oxide and nitric oxide synthase activity in proximal stumps of transected peripheral nerves. Neuroscience 91: 1515-1527, 1999[ISI][Medline].

44.   Zochodne, DW, Misra M, Cheng C, and Sun H. Inhibition of nitric oxide synthase enhances peripheral nerve regeneration in mice. Neurosci Lett 228: 71-74, 1997[ISI][Medline].


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