Inducible Nitric Oxide Synthase Expression in Brain Injury Cases

Background: The activity of Inducible Nitric Oxide Synthase (iNOS) is increased as a response to several insults to central nervous system causing brain injury. Objectives: To investigate iNOS expression in cadavers who died as result of traumatic brain injury (TBI), brain hemorrhage and brain congestion. Methodology: Indirect immunohistochemistry was performed on 80 cases of brain injury (autopsies), as well 23 normal brain cases as a control group. The cases were stained for iNOS and interpreted in terms of staining intensity from 0 to indicate negative, 1 weak, 2 moderate and 3 to indicate strong staining reactions. Results: personal variables (age, sex, brain weight) were not associated significantly with brain injury type. The iNOS was not express in control group. But, strong activity of iNOS was expressed in neurons astrocytes and in cases of TBI and brain hemorrhage. The activity of iNOS in blood vessels was moderate. The rate of expression of iNOS in congestion cases was low in comparison with TBI and hemorrhage cases. Conclusion: The activity of iNOS was shown to be significantly correlated with TBI and hemorrhage. Strong reactions were localized within neurons and astrocytes. This possibly explains the mediated effects of iNOS on brain blood vessels in terms of blood vessels dilatation to accommodate with the severity of brain injury cases.


Introduction
Nitric oxide synthase contributes through its product nitric oxide to transmission from one neuron to another, to the immune system and to dilating blood vessels. This enzyme has three isoforms, two are constitutive and the third is inducible. The Inducible Nitric Oxide Synthase (iNOS) is not detectable in normal brain condition [1]. It is an inflammatory product that is induced both in secondary damage [2] and in neurological recovery [3]. The inflammation response includes cytokine production, leucocytes infiltrations and microglial activation. A study reported that iNOS is not found in brain until 6-12 hours after Traumatic Brain Injury (TBI) [4].
The said enzyme is expressed in many different cells like epithelial cells, endothelial cells, macrophages and neurons [5]. A paper documented that iNOS has been detected in the vasculature, infiltrating macrophages and tumor cell of human brain [6].
While some researches demonstrated iNOS expression in primary astrocytes upon inflammatory induction as well as in neurological diseases whereas increase expression of iNOS sepsis, asthma, rheumatoid arthritis, atherosclerosis, tuberculosis and multiple sclerosis [7][8][9][10][11][12][13][14][15][16]. Hence it is clear that the activity of iNOS is increase as a result of several processes in Central Nervous System (CNS), causing brain injury. population having different environment, nutrition and lifestyle has been expressed particularly in the brain injury cases. Hence in the present attempt iNOS was investigated in the brain cadavers who died as result of traumatic brain injury, brain hemorrhage and brain congestion taken together (Figure 1-3), these observations suggest a relevant role of iNOS and its product NO in the inflammatory disease.

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Jet kind demonstrated that tumors derived from human colon Adenocarcinoma cell engineered to generate NO continuously [17].
They also descended that the exact of iNOS immunostaining was in related to progression increase in tumor [18]. Moreover, the role of iNOS is controversial, some experimental showed the beneficial role of enzyme, but in other contradicting findings were reported.
Hence for the work on the enzyme is needed on the all mentioned aspects to know it actual role. Then we can use its expression as a biological marker associate with brain injury cases in forensic medicine. subject who were not diagnosed using neuroprotective drugs or as brain injury cases or had multiple reasons for death were excluded. The age range of the study population was 2 to 90 years. These were 37 (46.2%) below the age of 30 and 43 (53.8%) above the age of 30. 23 cases of normal brain having same age grouped were also investigated as a control group. The attendant of all the cases were informed about the aim of the study and gave written consents for the investigation in accordance with ethical guideline.

Methodology
Tissue sample were obtained from cerebral cortex site of both control and study groups. Section from infected lung tissues known to express iNOS were processed as positive control. The paraffin tissue blocks were prepared, then cut on a freezing microtome into 5μm transverse sections and processed free-floating to make slide as per standard laboratory procedure. The slides were prepared and immersed for 05 minute first in acetone and then in vecta bond reagent according to instruction of vector laboratories USA. After that slides were washed with D.W, followed by drying in oven at 60°C for 30 minutes. Immunohistochemical detections of iNOS was performed using commercially available monoclonal mouse  (Table 1), iNOS staining reaction was more in neuron (58 cases), followed by astrocytes (51 cases) and blood vessels (45 cases). Negative staining was found more in blood vessels and less in neurons.  and study group (p<0.05). Among TBI, only 3 cases were negative, in neurons, astrocytes and blood vessels respectively. Hence the iNOS activity scored more in TBI and hemorrhage cases and less in congestion cases. The enzyme showed no association in expression with respect to age, sex and brain weight.

Discussion
As iNOS found only under inflammatory condition, that is why we focused our study to the iNOS expression in brain injury cases.
Our results highlighted that the changes occurred in the enzyme activity had no relation to age, sex and brain's weight. Besides, in normal human brain, the activity of enzyme was found NIL. Our findings are in consistent to the previous study who also found zero iNOS expression in normal colonic epithelium. We observed that iNOS activity in neurons was strong as compared to other sites in all types of brain injury cases. This is in line with other studies in which iNOS was expressed in epithelial cells, endothelial cells, macrophages, and neurons. The possible mechanism for the strong reaction may be that the action of NO often requires some other stimulation of target neurons before it can be effective. NO can also mediate the uptake and release of other neurotransmitters.
Moreover, NO blocks neurotransmitter receptors in brain injury cases. Second common staining was observed in astrocytes.
Like ours, several other studies also showed that iNOS activity is localized in astrocytes especially reactive astrocytes. One study described that NO attenuates astrocytic glutamate uptake and induces brain injury via effects on astrocyte function. This may be the reason that we found 90.5% positive cases in TBI and 70.4% in hemorrhage. One more study, also found that the induction of iNOS occurs predominantly in reactive astrocytes, our data revealed that blood vessels were not as affected by iNOS activity as neurons and astrocytes. iNOS has been reported to influence local cerebral blood flow. This effect is reported to be mediated through cGMP-gated ion channels.
In addition, contrasting findings have been found even regarding the cellular localization of iNOS within the tumor samples. Taken together, these observations suggest a relevant role of iNOS and its product NO in the inflammatory diseases. Jen kin et al. [17] demonstrated that tumors derived from human colon adenocarcinoma cells engineered to generate NO continuously.
They also described that the extent of iNOS immunostaining was in relation to progressive increase in tumor. Moreover, the role of iNOS is controversial; some experimental studies showed the beneficial role of enzyme, but in other contradicting findings were reported.
Hence further work on the enzyme is needed on all the mentioned aspects to know its actual role. After then we can use its expression as a biological marker associated with brain injury cases in forensic medicine.

Conclusion
The activity of iNOS was shown to be significantly correlated with TBI and hemorrhage. Strong reactions were localized within neurons and astrocytes. This possibly explains the mediated effects of iNOS on brain blood vessels in terms of blood vessels dilatation to accommodate with the severity of brain injury cases.