The activity of the Ang / Tie-2 system in the brain that suffered acute carbon monoxide poisoning

Abstract

Acute carbon monoxide poisoning (ACMP) leads to significant toxicity of the central nervous system and heart, and even death, following it, some patients suffered delayed encephalopathy. Until now, no theory had explained it exactly. It was reported that neovascularization was found in acute ischemic brains and also that angiopoietins (Ang) play important roles in the process of angiogenesis, for example, the members of Ang family, Ang-1 and Ang-2 may promote angiogenesis by combining with endothelial-specific cell surface tyrosine kinase receptor Tie-2. Interestingly, some studies suggested that small vascular injury may play an important role in the pathogenesis of delayed encephalopathy after carbon monoxide poisoning. Does neovascularization also occur in the brains after ACMP? Do Ang also take part in the pathologic processes in the brains that suffered ACMP? People know little about it. In the present study, we showed that neovascularization also occurred in the brains that suffered ACMP, and there are two expression peaks of Ang-1, Ang-2 and Tie-2, respectively, in the mice brains on the 3rd day and the 7th day following ACMP, and draw a conclusion that the Ang/Tie-2 system takes part in the pathologic processes in the brains that suffered ACMP by participating in neovascularization.

Introduction

Acute carbon monoxide poisoning (ACMP) is a common occupational poisoning with the highest morbidity and mortality, causing impairment in multi-system, mainly in the central nervous system. More unfortunately, following ACMP, some patients suffered a series of symptoms of encephalopathy known as delayed encephalopathy after carbon monoxide poisoning (DEACMP), manifesting as memory loss, impairment in extrapyramidal system or pyramidal system, often accompanied with disturbance of consciousness.

Although there are several hypotheses on the pathogenesis of DEACMP, such as hypoxia–ischemia, reperfusion injury and free radical formation, cytotoxic damage, the generation of excitatory amino acids, apoptosis and so on, ^{1 –4} none of them may explain it very well. Interestingly, the magnetic resonance imaging images of patients with DEACMP showed that there is extensive demyelination of white matter in the brain, ⁵ suggesting that small vascular injury may take part in the pathogenesis of DEACMP. Recently, it has been reported that neovascularization was found in the acute ischemic brain, ¹ however, people know little about the vascular changes in the brain that suffered ACMP.

It is known that angiopoietins (Ang) and vascular endothelial growth factor play important roles in the process of angiogenesis, specifically acting on endothelial cells, ⁶ and the members of Ang family, Ang-1 and Ang-2, closely related to angiogenesis, promoting angiogenesis by combining with endothelial-specific cell surface tyrosine kinase receptor Tie-2. ⁷ The Ang/Tie-2 system takes part in the final stage of angiogenesis, affecting the interaction of endothelial cells, smooth muscle cells and pericytes to promote angiogenesis and remodeling, so it may be a key regulator for angiogenesis in pathological states, ^{8 –13} for example ischemia. However, there is no report on the activity of Ang-1, Ang-2 and Tie-2 in the brain that suffered ACMP.

In the present study, we showed that neovascularization also occurred in the brain that suffered ACMP, and there are two expression peaks of the Ang-1, Ang-2 and Tie-2, respectively, in mice brains on the 3rd day and the 7th day following ACMP.

Material and methods

Animals

Animal experiments were performed after review and approval by the Chinese government according to the local committee for animal studies and established standards for human handling. Healthy ICR male mice were divided into the air control group and the ACMP group, 48 mice in each group. Each group was divided into eight subgroups according to the following different time points: 6 h, l d, 2 d, 3 d, 4 d, 7 d, 14 d and 28 d, 6 mice in each subgroup. Animal care and experimental protocols were approved by Dalian Medical University, China. At each indicated time point, animals were killed, then brain tissue was obtained and sectioned for immunohistochemistry assay and reverse transcription polymerase chain reaction (RT-PCR) assay.

Mouse model of ACMP

Each mouse in the ACMP group was given a single intraperitoneal injection of 90% median lethal dose, 99.9% carbon monoxide gas (170 ml/kg), and each mouse in the air control group was given the same amount of air by intraperitoneal injection. ¹⁴

Reverse transcription polymerase chain reaction

Total RNA from the whole brain of the mouse in each subgroup was extracted using a Trizol reagent (Invitrogen, California, USA) and reverse transcribed with moloney murine leukemia virus reverse transcriptase (Invitrogen). Polymerase chain reaction analysis was performed in a final volume of 20 μl using PCR Promega (Madison, Wisconsin, USA). ¹⁵ Mouse Ang-1-specific primer sequence (forward: 5′-GGAGCATGTGATGGAAAATTA-3′; reverse: 5′-TGTGTTTTCCCTCC ATTTCTA-3′), mouse Ang-2-specific primer sequence (forward: 5′- AAAGAGT ACAAAGAGGGCT TC-3′; reverse: 5′-TCCAGTAGTACCACTTGATAC-3′), mouse Tie-2-specific primer sequence (forward: 5′-ATGGACTCTTTAGCCGGCTTA-3′; reverse: 5′-CCTTATAG CCTGTCCTCGAA-3′) and mouse β-actin-specific primer sequence (forward: 5′-TCATC ACATTTGGCAACGAGC-3′, reverse: 5′-AACA GTCCGCCTAG AAGCAC-3′) were used.

Histology assay and immunohistochemistry assay

After killing the mice, the brains were obtained and made into paraffin tissue blocks. Then the paraffin tissue blocks were submitted into 5 μm-thick serial cross sections. Of the two consecutive sections, one was destined for histological assay and the other for immunohistochemical analysis. The paraffin-embedded tissue sections were first deparaffinized and hydrated in xylene and graded alcohol series. Then, antigen retrieval was performed using microwave treatment in citrate-buffer (10 mM, pH 6.0), and endogenous peroxidase activity was blocked with 3% hydrogen peroxide/methanol. Thereafter, sections were blocked with solution containing 10% bovine serum (DakoCytomation, Glostrup, Denmark) for 45 min. After that, the histology slides were stained with hematoxylin, eosin, Orange G and alcian blue (H&E); the immunohistochemistry slides were incubated with primary antibody (rabbit anti-mouse Ang-1, Ang-2 or Tie-2 polyclonal, 1:100, Sanata, Texas, USA) overnight at 4°C. Primary antibody was detected after incubation with a biotinylated secondary antibody (biotinylated goat anti-rabbit IgG, 1:1,Wuhan Boster, Wuhan Hubei, China) using the Vectastain Elite ABC Kit (Vector Laboratories, California, USA) and the FAST DAB Tablet Set (Sigma, St. Louis, USA). Sections were counterstained with Meyer’s Hemalaun and mounted with Pertex. ¹⁶

The histology slides with typical hippocampus region were selected (referring to The Mouse Brain in Stereotaxic Coordinates ¹⁷ ), and the microvessels with lumen whose lamina muscularis were not formed in five randomly selected visual fields at the hippocampus region on each histology slide were counted, respectively, with images recorded in a Leica DMLB microscope (Leica, Bensheim, Germany) using 200× lens, and the mean value was calculated.

The determination of labeling levels for each antibody was performed semiquantitatively. The immunohistochemistry slides with typical hippocampus region were selected, and five visual fields (200×) at the hippocampus region on each immunohistochemistry slide were selected randomly to score synthetically, according to color intensity and grade of positive cell number: A represented the grade of positive cell number, using scores from 0 to 4 (0 = 0–1%, 1 = 1–10%, 2 = 10–50%, 3 = 50–80%, 4 = 80–100%); B represented the color intensity of positive cells, using scores from 0 to 3, colorless = 0 (negative), pale yellow = 1 (weakly positive), brownish yellow = 2 (positive) and brownish tan = 3 (strongly positive); then, IHS (Intensity Hue Saturation (IHS) = A × B) is used to calculate the rate of positive expression and the mean value was calculated too. ¹⁸

Statistical analysis

All data are expressed as mean ± SD. Student’s t test for two groups or one-way analysis of variance and post hoc multiple comparisons (Least significant difference (LSD) test) for three or four groups were performed to evaluate the statistical significance using the SPSS 13.0 statistical software package (SPSS, Inc., Chicago, IL, USA). Tests were two sided, and p < 0.05 was considered as statistically significant.

Results

The changes in microvessel count and pathological morphous

To investigate the pathological changes of brain after ACMP, we performed H&E staining on the mice brain sections at different time points after ACMP and observed at the hippocampus region with microscope. The results showed that, compared with the air control group, on the 6th h after ACMP, there are more edematous brain tissues and swollen neurons, with the gap around neurons and blood vasculars increasing greatly. From the 3rd day after poisoning, in addition to the above changes, the cells presented ischemia, degeneration and necrosis. The edema was first observed at the hippocampus region in the brain on the 6th h after poisoning, and peaked on the fourth day and the 7th day after poisoning, declined gradually till the 21st day after poisoning, in addition on the 28th day after poisoning, there are still a part of necrotic cells in the injured hippocampus tissue (Figure 1).

Figure 1.

The histologic photomicrography featuring the brain tissue. (a) The histologic assay showing the brain tissue; scale bar, 50 μm. (b) Quantification of the microvessel in the brain tissue, microvessels with lumen whose lamina muscularis is unformed in each histology slide were counted, the carbon monoxide poisoning group (CO group) versus the air control group (Con group) at each time point, **p < 0.01,***p < 0.001, Student’s t test, n = 5 in each group.

A small amount of irregular micrangiums was first observed at the hippocampus region in the brain on the 6th h after poisoning, and peaked on the 14th day after poisoning; on the 21st day after poisoning, there are lots of cord-like vascular segments at the hippocampus region in the brain, with lumens expanding; until the 28th day after poisoning, microvascular segments at the hippocampus region in the brain decreased greatly (p < 0.05). There were statistically significant differences in microvessel count at the hippocampus region in the brains between the ACMP group and the control group from the 3rd day to the 28th day after poisoning (Figure 1(a) and (b); p < 0.01).

Bimodal increase in the transcriptional level of Ang-1, Ang-2 and Tie-2 in the mice brains that suffered ACMP

To investigate the activity of Ang-1, Ang-2 and Tie in the mice brains after ACMP, total RNA from the mice brains was extracted at different stages after ACMP and the transcriptional levels of Ang-1, Ang-2 and Tie were, respectively, measured by RT-PCR assay (Figure 2). The results showed that the transcriptional levels of Ang-1 in the mice brains of the ACMP group were significantly higher than the level in the control group at different time points (Figure 2(a) and (b); p < 0.001). After poisoning, it increased gradually and peaked on the 3rd day, after that, it declined gradually till the 7th day and formed the second transcriptional peak, then it declined again and maintained at the lower level on the 28th day (p < 0.05).

Figure 2.

The transcriptional level of the Ang/Tie-2 system. (a) RT-PCR assay showing mRNA levels of Ang-1 in mice brains in each subgroup at each time point, the CO group versus the Con group at each time point, ***p < 0.001, Student’s t test, n = 6 in each group. (b) RT-PCR assay showing mRNA levels of Ang-2 in mice brains in each subgroup at each time point, the CO group versus the Con group at each time point, **p < 0.01, ***p < 0.001, Student’s t test, n = 6 in each group. (c) RT-PCR assay showing mRNA levels of Tie-2 in mice brains in each subgroup at each time point, the CO group versus the Con group at each time point, ***p < 0.001, Student’s t test, n = 6 in each group. (d) RT-PCR assay showing mRNA levels of β-actin in mice brains in each subgroup at each time point. RT-PCR: reverse transcription–polymerase chain reaction.

The transcriptional level of Ang-2 in the mice brains that suffered ACMP showed a similar tendency to Ang-1: its first peak appeared on the 3rd day, and the second peak appeared at the 7th day after poisoning (Figure 2(c) and (d); p < 0.05). Tie-2 in the mice brains also showed bimodal increase in the transcriptional level on the 3rd day and the 7th day after poisoning as Ang-1 (Figure 2(e) and (f); p < 0.05). Both the transcriptional levels of Ang-2 and Tie-2 in the mice brains of the ACMP group were significantly higher than the levels in the control group at different time points (Figure 2(e) and (f); p < 0.001).

Bimodal increase in expression of Ang-1, Ang-2 and Tie-2 at the hippocampus in the mice brains that suffered ACMP

To investigate the activity of Ang-1, Ang-2 and Tie-2 at the hippocampus in the mice brains after ACMP at different stages, the expression levels of Ang-1, Ang-2 and Tie-2 at the hippocampus were, respectively, measured by immunohistochemistry assay (Figure 3(a), 4(a) and 5(a)). The results showed that the expression level of Ang-1 at the hippocampus in the mice brains in the ACMP group is significantly higher than the level in the control group (p < 0.001). After poisoning, it increased gradually and peaked on the 3rd day, after that, it declined gradually till the 7th day and formed the second expression peak, then declined again and maintained at the lower level on the 28th day (Figure 3(b), p < 0.05).

Figure 3.

The expression level of Ang-1. (a) Immunohistochemistry assay showing the expression levels of Ang-1 in mice brains in each subgroup at each time point; scale bar, 50 μm. (b) Quantification of the expression of Ang-1 by scoring the color intensity and grade of positive cell number, the CO group versus the Con group at each time point, ***p < 0.001, Student’s t test, n = 6 in each group.

The expression of Ang-2 at the hippocampus in the mice brains showed a similar tendency to Ang-1 after poisoning (Figure 4, p < 0.05). The Tie-2 at the hippocampus in the mice brains also showed bimodal increase in expression on the 3rd day and the 7th day after poisoning as Ang-1 (Figure 5, p < 0.05). Both the expression levels of Ang-2 and Tie-2 in the mice brains of the ACMP group were significantly higher than the levels in the control group at different time points (Figure 4, p < 0.001; Figure 5, p < 0.01).

Figure 4.

The expression level of Ang-2. (a) Immunohistochemistry assay showing the expression levels of Ang-2 in mice brains in each subgroup at each time point; scale bar, 50 μm. (b) Quantification of the expression of Ang-2 by scoring the color intensity and grade of positive cell number, the CO group versus the Con group at each time point, ***p < 0.001, Student’s t test, n = 6 in each group.

Figure 5.

The expression level of Tie-2. (a) Immunohistochemistry assay showing the expression levels of Tie-2 in mice brains in each subgroup at each time point; scale bar, 50 μm. (b) Quantification of the expression of Tie-2 by scoring the color intensity and grade of positive cell number, the CO group versus the Con group at each time point, **p < 0.01, Student’s t test, n = 6 in each group.

Discussion

In the present study, we showed that there are a great number of buckling microvascular segments in irregular shape at the hippocampus in the mice brains on the 3rd day after ACMP. It seemed that lots of neoformative micrangium formed at this region, and the angiogenesis peaked on the 14th day after ACMP. On the 28th day after ACMP, micrangium become matured, with part of micrangium segment extincting, suggesting that neovascularization does appear in the brains that suffered ACMP.

Our results also showed that the transcriptional level of Ang-1 in the mice brains first peaked on the 3rd day after ACMP, then formed the second peak at the 7th day after poisoning, after that it declined again but still statistically higher than the level in the control group; the transcription of Ang-2 in the mice brains showed a similar tendency to Ang-1 after poisoning; Tie-2 in the mice brains also showed bimodal increasing transcription on the 3rd day and the 7th day after poisoning as Ang-2. The further study also showed that there is bimodal increase in expression of Ang-1, Ang-2 and Tie-2 at the protein level in the mice brains after ACMP.

Our data showed that, both at the mRNA level and at the protein level, Ang-1, Ang-2 and Tie-2 manifested as two upregulated phases after ACMP, different from the one expression peak in focal cerebral ischemia. It is known that the upregulation of Ang-1 may reduce the vascular endothelial permeability ¹⁹ to alleviate cerebral edema, and Ang-2 may damage vascular stability to loose the connection between extracellular matrix, supporting vascular endothelial cells and other kinds of cells by antagonizing Ang-1 to bind Tie-2 receptor, contributing to neovascularization and budding. ²⁰ The first upregulation of the Ang/Tie-2 system after ACMP may be explained as that the acute hypoxia and ischemia of brain tissue led to the release of a large number of free radicals, while an effective collateral circulation was established in order to improve the state of cerebral ischemia and hypoxia, then the Ang/Tie-2 system was on the emergence of a compensatory upregulation, participating in angiogenesis together with other vascular-specific factors. After that, the ischemia and hypoxia of brain caused by carbon monoxide poisoning were improved after the mouse breaking away from the poisoning environment, so the expression of the Ang/Tie-2 system began to drop down; however, the brain injury caused by ischemia and hypoxia still persists after ACMP, the Ang/Tie-2 system expression level is higher than normal levels.

However, the specific causes and mechanisms of the second upregulation of the Ang/Tie-2 system after ACMP are not clear. We had showed that the brain tissue edema was first observed on the 6th h after poisoning, and peaked on the 4th day and the 7th day after poisoning; we speculated that, at the two time points, the cerebral ischemia and hypoxia increased greatly, meanwhile, carbon monoxide poisoning may affect multiple systems and lead to some unknown mechanisms followed by potential confounding factors, contributing to the occurrence of DEACMP. While the Ang/Tie-2 system compensatory increased again on the 7th day after ACMP to antagonize the increasing apoptosis of endothelial cells, induce sprouting of new blood vessels, maintain the integrity of the vessel wall, reduce vascular permeability and cerebral edema, meanwhile, protect neurons and restore the function of brain tissue.

However, the present study suffers from several limitations. For example, in this study we only showed the activity of the Ang/Tie-2 system in the brain after ACMP, while we know little about the changes in the Ang/Tie-2 system in the brain that suffered DEACMP. The change in microvessel count and pathological morphous in the brain that suffered DEACMP also need more study.

Notwithstanding these limitations, this study showed that there are bimodal upregulation of Ang-1, Ang-2 and Tie-2 in the mice brains after ACMP, suggesting that the second upregulation of the Ang/Tie-2 system may be related to DEACMP by participating in the reconstruction of cerebral microvessels. Then, we draw a conclusion that the Ang/Tie-2 system takes part in the pathologic processes in the brains that suffered ACMP by participating in neovascularization.