Inhibition of nitric oxide synthase aggravates brain injury in diabetic rats with traumatic brain injury

Abstract

Studies have shown that hyperglycemia aggravates brain damage by affecting vascular endothelial function. However, the precise mechanism remains unclear. Male Sprague-Dawley rat models of diabetes were established by a high-fat diet combined with an intraperitoneal injection of streptozotocin. Rat models of traumatic brain injury were established using the fluid percussion method. Compared with traumatic brain injury rats without diabetic, diabetic rats with traumatic brain injury exhibited more severe brain injury, manifested as increased brain water content and blood-brain barrier permeability, the upregulation of heme oxygenase-1, myeloperoxidase, and Bax, the downregulation of occludin, zona-occludens 1, and Bcl-2 in the penumbra, and reduced modified neurological severity scores. The intraperitoneal injection of a nitric oxide synthase inhibitor N(5)-(1-iminoethyl)-L-ornithine (10 mg/kg) 15 minutes before brain injury aggravated the injury. These findings suggested that nitric oxide synthase plays an important role in the maintenance of cerebral microcirculation, including anti-inflammatory, anti-oxidative stress, and anti-apoptotic activities in diabetic rats with traumatic brain injury. The experimental protocols were approved by the Institutional Animal Care Committee of Harbin Medical University, China (approval No. ky2017-126) on March 6, 2017.

Keywords: apoptosis; blood-brain barrier; brain edema; diabetes mellitus; inflammation; injury; neurological function; nitric oxide synthase; traumatic brain injury.

Figure 1

A diagram illustrating the protocol and changes in brain water contents in rats with DM complicated with TBI. (A) A diagram illustrating the protocol. The bottom horizontal axis represents time, and the arrows indicate the experimental operation performed at each time. (B) Water contents of damaged ipsilateral brain tissues, 48 hours after TBI, detected by dry-wet weight method. Brain water content (%) = (wet weight − dry weight)/(wet weight) × 100. Data are expressed as the mean ± SD (n = 6). *P < 0.05 (one-way analysis of variance followed by Bonferroni post hoc test). BBB: Blood-brain barrier; DM: diabetes mellitus; L-NIO: N(5)-(1-iminoethyl)-L-ornithine; mNSS: modified neurological severity score; TBI: traumatic brain injury.

Figure 2

Changes in the blood-brain barrier permeability in the injured penumbra and hippocampus of rats with DM complicated with TBI. The blood-brain barrier permeability was evaluated by Evans blue staining. (A) The permeability of Evans blue dye in rat brain tissues. Compared with the Sham group, rats in the TBI group demonstrated an increased Evans blue penetration rate on the injured side of the brain, 2 days after surgery. Hyperglycemia significantly increased the penetration rate of Evans blue in the injured brain tissue. (B) Quantitative results of sodium fluorescein levels. Data are expressed as the mean ± SD (n = 5). *P < 0.05 (one-way analysis of variance followed by Bonferroni post hoc test). DM: Diabetes mellitus; L-NIO: N(5)-(1-iminoethyl)-L-ornithine; RFU: relative fluorescence unit; TBI: traumatic brain injury.

Figure 3

Pathological changes in the penumbra region of rats with DM complicated with TBI. Hematoxylin and eosin (HE) staining and Nissl staining in brain tissues from the rats in the four experimental groups (n = 6). The results showed that the brain tissue of rats in the Sham group displayed a regular cellular morphology, with uniform distribution, clear nucleoli and borders, rich cytoplasm, and no obvious neuronal degeneration and necrosis. The brain tissues from the other three groups of rats showed varying degrees of brain tissue destruction, massive hemorrhage, tissue relaxation and edema, and decreased cytoplasmic neighbors. Arrows indicate pathological changes. Scale bars: 400 µm and 40 µm (as labeled). DM: Diabetes mellitus; HE: hematoxylin-eosin; L-NIO: N(5)-(1-iminoethyl)-L-ornithine; TBI: traumatic brain injury.

Figure 4

Expression levels of proteins associated with the blood-brain barrier and the regulation of inflammation-related protein in the penumbra area of rats with DM complicated with TBI. (A–E) Immunopositivity (arrows) of HO-1, MPO, occludin, and ZO-1 proteins (A) and the percentage of positive cells (B–E). The results showed that compared with those in the other groups, the expression levels of HO-1 and MPO in the TBI + DM + L-NIO group increased significantly, whereas the expression levels of ZO-1 and occludin decreased significantly. Scale bars: 400 µm and 40 µm (as labeled). Data are expressed as the mean ± SD (n = 6). *P < 0.05 (one-way analysis of variance followed by Bonferroni post hoc test). DM: Diabetes mellitus; HO-1: heme oxygenase-1; L-NIO: N(5)-(1-iminoethyl)-L-ornithine; MPO: myeloperoxidase; TBI: traumatic brain injury; ZO-1: zonula occludens-1.

Figure 5

Protein expression levels of Bax, Bcl-2, occludin, and MPO in the penumbra area of rats with DM complicated with TBI. (A) The detection of Bax, Bcl-2, occludin, and MPO protein expression by western blot. (B) Quantification of Bax, Bcl-2, occludin, and MPO protein expression levels. Data are expressed as the mean ± SD (n =5). *P < 0.05 (one-way analysis of variance followed by Bonferroni post hoc test). DM: Diabetes mellitus; L-NIO: N(5)-(1-iminoethyl)-L-ornithine; MPO: myeloperoxidase; TBI: traumatic brain injury.