J Korean Med Sci.  2005 Aug;20(4):663-669. 10.3346/jkms.2005.20.4.663.

The Role of Inducible Nitric Oxide Synthase Following Spinal Cord Injury in Rat

• Kwak EK
• Kim JW
• Kang KS
• Lee YH
• Hua QH
• Park TI
• Park JY
• Sohn YK

• KMID: 1712751
• DOI: http://doi.org/10.3346/jkms.2005.20.4.663

Abstract

Acute spinal cord injury (SCI) is two-step process that first involves the primary mechanical injury and then the secondary injury is induced by various biochemical reactions. Apoptosis is one of secondary SCI mechanisms and it is thought to play an important role for the delayed neuronal injury. The enhanced formation of nitric oxide (NO) via inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of apoptosis in SCI. The level of .iNOS mRNA peaked at 6 hr after SCI and it declined until 72 hr after SCI in a rat model. Double-immunofluorescence staining revealed that iNOS positive cells were stained for ED-1, synaptophysin, GFAP, and oligodendrocyte marker. The terminal deoxynucleotidyl-transferase-mediated dUDP-biotin nick end-labeling (TUNEL) positive cell count was higher for the 72 hr post-SCI group than for the 24 hr post-SCI group. This cell count was also higher going in the caudal direction than in the rostral direction from the epicenter, and especially for the 72 hr group. Treatment with a selective iNOS inhibitor resulted in the reduction of TUNEL-positive cells at the lesion site. These findings suggest that nitric oxide generated by the iNOS of macrophages, neurons, oligodentrocytes, and astrocytes plays an important role for the acute secondary SCI that results from apoptotic cell death.

Keyword

inducible nitric oxide synthase; Spinal Cord Injuries; Apoptosis; Rats

MeSH Terms

Analysis of Variance
Animals
Apoptosis
Comparative Study
Glial Fibrillary Acidic Protein/analysis
In Situ Nick-End Labeling
Microscopy, Fluorescence
RNA, Messenger/genetics/metabolism
Rats
Rats, Sprague-Dawley
Research Support, Non-U.S. Gov't
Reverse Transcriptase Polymerase Chain Reaction
Spinal Cord/chemistry/enzymology/pathology
Spinal Cord Injuries/*enzymology/pathology/physiopathology
Time Factors

Figure

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  Fig. 1 iNOS mRNA expression by real time reverse transcription-polymerase chain reaction (RT-PCR) in the control group and 6 hr, 24 hr, 72 hr after contusion injury. The relative amounts of iNOS mRNA shows noticeable changes with time. At 6 hr post-SCI, the increase in the level of iNOS mRNA was prominent, especially in the caudal side.

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  Fig. 2 nNOS mRNA expression by real time RT-PCR in the control group and at 6 hr, 24 hr, and 72 hr after contusion injury. The relative amounts of nNOS mRNA showed no noticeable change with time, and were much the same in the control and experimental groups.

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  Fig. 3 The terminal deoxynucleotidyl-transferase-mediated dUDP-biotin nick end-labeling (TUNEL)-positive cell count was reduced after iNOS inhibition 72 hr after spinal cord injury (p<0.05). 6 hr=6 hr after SCI before iNOS inhibition, 6 hr (SMT)=6 hr after SCI after iNOS inhibition, 72 hr=72 hr after SCI before iNOS inhibition, 72 hr (SMT)=72 hr after SCI after iNOS inhibition.

• 

  Fig. 4 The terminal deoxynucleotidyl-transferase-mediated dUDP-biotin nick end-labeling (TUNEL)-positive cell count was higher at 72 hr post-SCI than at 24 hr post-SCI and higher in the caudal side than in the rostral side. No statistical significance could be attached to different of TUNEL-positive cell count in the gray matter and white matter (p<0.05).

• 

  Fig. 5 Immunofluorescence of iNOS in the rat spinal cord of the control (A, ×200), and in the 72 hr group (B, ×200). In the control group, iNOS activity is absent. In the experimental group, iNOS activity appeared at 24 hr and increased in intensity at 72 hr.

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  Fig. 6 Immunofluorescence of nNOS in the rat spinal cord of the control (A, ×200), and of the 72 hr group (B, ×200), 72 hr after spinal cord contusion. nNOS activity was observed in both the control and experimental groups. In the experimental group, nNOS immunoreactivity is observed in the neuronal cell body 24 hr post-SCI and in the cell processes at 72 hr post-SCI.

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  Fig. 7 Double labeling of iNOS with cell markers in rat spinal cord. (A, D) iNOS-immunoreactive cells, (B) microglia marker (ED-1-immunoreactive cells), (E) oligodendrocyte marker (rat oligodendrocyte monoclonal antibody-immunoreactive cells), (C, F) merged. (×400).

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