Hyperhomocysteinemia-Induced Oxidative Stress Exacerbates Cortical Traumatic Brain Injury Outcomes in Rats

Abstract

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality among military service members and civilians in the United States. Despite significant advances in the understanding of TBI pathophysiology, several clinical reports indicate that multiple genetic and epigenetic factors can influence outcome. Homocysteine (HCY) is a non-proteinogenic amino acid, the catabolism of which can be dysregulated by stress, lifestyle, aging, or genetic abnormalities leading to hyperhomocysteinemia (HHCY). HHCY is a neurotoxic condition and a risk factor for multiple neurological and cardiovascular disorders that occurs when HCY levels is clinically > 15 µM. Although the deleterious impact of HHCY has been studied in human and animal models of neurological disorders such as stroke, Alzheimer's disease and Parkinson's disease, it has not been addressed in TBI models. This study tested the hypothesis that HHCY has detrimental effects on TBI pathophysiology. Moderate HHCY was induced in adult male Sprague Dawley rats via daily administration of methionine followed by impact-induced traumatic brain injury. In this model, HHCY increased oxidative stress, upregulated expression of proteins that promote blood coagulation, exacerbated TBI-associated blood-brain barrier dysfunction and promoted the infiltration of inflammatory cells into the cortex. We also observed an increase of brain injury-induced lesion size and aggravated anxiety-like behavior. These findings show that moderate HHCY exacerbates TBI outcomes and suggest that HCY catabolic dysregulation may be a significant biological variable that could contribute to TBI pathophysiology heterogeneity.

Keywords: Heterogeneity; Hyperhomocysteinemia; Pathophysiology; Stress; Traumatic-brain injury.

Fig. 1

Hyperhomocysteinemia induction by systemic methionine. a Methionine injections, once daily, to CCI and Sham-operated rats at 300 mg/kg sustained a six-fold increase in homocysteine levels in both Sham and CCI groups compared to saline-injected rats (n = 10/group; ***p < 0.001). b Hyperhomocysteinemia had no significant effect on the percent weight gain on both sham-operated and CCI rats (n = 10/group; p > 0.05). Results are graphed with individual HCY values and standard deviation with 95% CV

Fig. 2

Cerebral cortex lipid peroxidation is elevated by hyperhomocysteinemia. a Immunoblots illustrating the expression of 4-hydroxynonal in the S1/S2 cortex at days 1 and 7 post-surgery. b Quantification of protein bands indicated that 4-HNE expression was significantly increased in the cortex HHCY-CCI rats than NHCY-sham and CCI at days 1 or 7 post-surgery (n = 5; ^#,&p < 0.05 and **p < 0.01). Changes in 4-HNE expression were also significantly greater in the cortex HHCY-sham rats than NHCY-shams

Fig. 3

Expression of nitrotyrosine in the corpus callosum of rats subjected to traumatic brain injury and potentiation by hyperhomocysteinemia. a Representative fluorescent images demonstrating the presence of nitrotyrosine (green) and DAPI-stained nuclei (blue) in rat corpus callosum b Quantitation of nitrotyrosine positive cells indicated that nitrotyrosine expression was significantly increased in the corpus callosum of CCI-injured NHCY rats 24 h post-injury compared NHCY sham (n = 5; *^&,#p < 0.05). Moreover, nitrotyrosine expression in the corpus callosum was further potentiated by hyperhomocysteinemia (n = 5; **p < 0.01)

Fig. 4

Decreased expression of Zona occludens 1 and occludin in the cortex following TBI and exacerbation by hyperhomocysteinemia. a Immunoblots depicting the expression of endothelial tight junction proteins ZO-1 and occludin in rat’sS1/S2 cortex. Quantitation of protein band intensities b showed post-trauma time-dependent variation in cortical expression of ZO-1. ZO-1 expression was significantly decreased in HHCY-CCI rat cortex on day 7-post-injury compared to HHCY-sham and NHCY-CCI rats at day 1 pi (n = 5; ^#p < 0.05). Compared to NHCY-sham rats, ZO-1 expression was also significantly reduced at day 7 post-injury in NHCY rats and at day 1 post-surgery in HHCY-sham rats (n = 5; *p < 0.05, ***p < 0.001). By contrast, c occludin expression was only affected by HHCY and not CCI injury and was significantly decreased in the cortex of HHCY-sham and CCI rats (n = 5; *^,#p < 0.05)

Fig. 5

Hyperhomocysteinemia exacerbates Evans blue extravasation following TBI. a Representative brain images from NHCY and HHCY-sham and -CCI rats injected with Evans blue solution at days 1 or 7 post-surgery. b Quantitation of Evans blue in formamide showed a significant increase in Evans blue extravasation in the ipsilateral hemisphere of NHCY-CCI rats at day 1 post-injury (n = 5/group; *p < 0.05 and **p < 0.01). This effect was aggravated by homocysteine accumulation. Despite the reduction of the amount of HCY in the brain parenchyma, the detrimental effect of HHCY persisted, till day 7 post-surgery (n = 5/group; ^#p < 0.05 and ^##p < 0.01)

Fig. 6

Increased thrombin expression by hyperhomocysteinemia in rat plasma. a Representative immunoblots illustrating the expression of thrombin in rat plasma. Quantitation of protein band intensity b showed increased thrombin expression both in HHCY-sham and -CCI rats on day 1 and 7 post-surgery compared to NHCY counterparts (n = 6/group; ^#p < 0.05, ***p < 0.001)

Fig. 7

Aggravation of TBI-induced von Willebrand factor expression by hyperhomocysteinemia. a Representative microscopic images showing the expression of von Willebrand factor in rat cortex at day 1 post-surgery. b The quantitation of the percent area of von Willebrand factor immunoreactivity indicated that its expression was induced by CCI at day 1 post-injury compared to NHCY-Sham rats (n = 5; ^#p < 0.05). That effect was intensified by HHCY (n = 5; **p < 0.01)

Fig. 8

Homocysteine accumulation enhances ICAM-1 expression after TBI. a Representative microscopic images showing ICAM-1 (green) and laminin (red) and DAPI-stained nuclei (blue) in rats somatosensory 1 and 2 cortices at day 7 post-surgery. b Quantitation showed that the percent area covered by ICAM-1 expressing cells was significantly higher in HHCY-CCI rat brain compared to shams and NHCY-CCI rats (n = 5; *p < 0.05, **p < 0.01)

Fig. 9

Hyperhomocysteinemia causes increased and diffuse presence of inflammatory cells in the rat brain parenchyma after TBI. a Representative microscopic images showing CD-68 immuno-positive cells in rats somatosensory 1 and 2 cortices and the corpus callosum at day 7 post-surgery. b Quantitation of the percent CD-68 immuno-positive brain area indicated that CCI injury caused a significant infiltration of inflammatory cells in the rat brain and that condition was exacerbated by HCY accumulation resulting in significantly greater area coverage by CD-68 positive cells (n = 5, *p < 0.05, **^,##p < 0.01 and ***p < 0.001)

Fig. 10

Hyperhomocysteinemia worsens TBI-induced cortical lesion volume. a Representative hematoxylin and eosin eosin-stained coronal brain sections showing cortical impact-induced brain lesion. b Lesion volume quantitation showed that HHCY aggravated CCI-induced cortical brain lesion compared (n = 5; *p < 0.05), compared to sham rats

Fig. 11

Anxiety-like behavior is worsened by hyperhomocysteinemia plus TBI. a Time spent by animals in the open arms of the Elevated Plus maze at day 2, 8, 15 and 29 days-pi. Hyperhomocysteinemia caused significant reduction of the time spent by CCI rats exploring the open arms of the maze at days 3 and 9 post-surgery compared to NHCY-shams (n = 11; ^#p < 0.05) and NHCY-CCI rats (n = 11; ^#p < 0.05). b The total distance traveled by rats in the maze was not different among rats in different experimental groups (n = 11; p > 0.05)