VCAM1 Drives Vascular Inflammation Leading to Continuous Cortical Neuronal Loss Following Chronic Cerebral Hypoperfusion

Abstract

Background:

Chronic cerebral hypoperfusion (CCH) is associated with neuronal loss and blood-brain barrier (BBB) impairment in vascular dementia (VaD). However, the relationship and the molecular mechanisms between BBB dysfunction and neuronal loss remain elusive.

Objective:

We explored the reasons for neuron loss following CCH.

Methods:

Using permanent bilateral common carotid artery occlusion (2VO) rat model, we observed the pathological changes of cortical neurons and BBB in the sham group as well as rats 3d, 7d, 14d and 28d post 2VO. In order to further explore the factors influencing neuron loss following CCH with regard to cortical blood vessels, we extracted cortical brain microvessels at five time points for transcriptome sequencing. Finally, integrin receptor a4β1 (VLA-4) inhibitor was injected into the tail vein, and cortical neuron loss was detected again.

Results:

We found that cortical neuron loss following CCH is a continuous process, but damage to the BBB is acute and transient. Results of cortical microvessel transcriptome analysis showed that biological processes related to vascular inflammation mainly occurred in the chronic phase. Meanwhile, cell adhesion molecules, cytokine-cytokine receptor interaction were significantly changed at this phase. Among them, the adhesion molecule VCAM1 plays an important role. Using VLA-4 inhibitor to block VCAM1-VLA-4 interaction, cortical neuron damage was ameliorated at 14d post 2VO.

Conclusion:

Injury of the BBB may not be the main reason for persistent loss of cortical neurons following CCH. The continuous inflammatory response within blood vessels maybe an important factor in the continuous loss of cortical neurons following CCH.

Keywords

Blood-brain barrier chronic cerebral hypoperfusion inflammation neuron VCAM1

INTRODUCTION

Vascular dementia (VaD) is the second most common dementia subtype in older adults. There is substantial evidence that patients with VaD have some degree of chronic cerebral hypoperfusion (CCH) [1, 2], and CCH can be considered one of the most important risk factors for VaD. Progressive cognitive decline is the main manifestations of VaD, which is attributable to neuronal loss. CCH precedes the formation of brain damage and cognitive impairment in VaD [3].

During CCH, cerebral blood flow is gradually reduced, leading to cognitive impairment and neurodegenerative disease. CCH-induced progressive brain parenchymal damage comprises metabolic disturbances, white matter damage, neuronal and pyroptotic apoptosis, impaired synaptic plasticity, activated neuroinflammation, and neuroendocrine disturbances [4]. Because of this association, determining how CCH modulates the cellular processes underlying parenchymal lesion formation is critical for understanding the pathophysiology, temporal progression, and potential clinical implications of VaD.

There is a series of pathological changes proceeding from CCH and leading to these brain injuries, the most studied of which is damage to the blood-brain-barrier (BBB). The BBB is composed mainly of capillary endothelial cells (ECs) that are fused by tight junctions [5 –9]. The maintenance of the barrier function falls on the interaction between the ECs, the astrocytic foot processes and pericytes. And BBB establishes a controlled environment in the brain parenchyma by maintaining relatively constant levels of hormones, nutrients, and water in the brain. Any fluctuation in the brain microenvironment may disrupt the homeostasis of the central nervous system, resulting in damage to the brain parenchyma.

However, in animal models of CCH, increased BBB permeability occurs acutely, rather than as a persistent process. Furthermore, the most pronounced BBB damage occurs in the early stages of injury [10]. However, brain damage usually occurs in the chronic phase following CCH [11]. Changes in BBB injury and neuronal injury following CCH are mutually inconsistent. Previous studies have also suggested that reduced functional integrity of the BBB precedes neuronal damage in many CNS diseases [12 –17].

White matter lesions are the main pathological manifestation of injury caused by CCH [18, 19], but the loss of cortical neurons is also an inevitable pathological progression of CCH [20]. CCH causes progressive cognitive impairment due to disturbance of the circulatory system. Therefore, vascular changes and neuronal damage in the cortex following CCH are also worthy of attention.

In this study, we used permanent bilateral common carotid artery occlusion (2VO) rat model to observe the pathological changes of cortical neurons and BBB in the sham group as well as rats 3d, 7d, 14d and 28d post 2VO. We further extracted cortical brain microvessels at five time points for transcriptome sequencing to explore the factors influencing neuron loss following CCH with regard to cortical blood vessels. Our results indicate that injury of the BBB may not be the main reason for persistent loss of cortical neurons following CCH. Results of cortical microvessel transcriptome analysis showed that biological processes related to vascular inflammation mainly occurred in the chronic phase. Meanwhile, cell adhesion molecules, cytokine-cytokine receptor interaction were significantly changed at this phase. Among them, the adhesion molecule VCAM1 plays an important role. VCAM1, a member of the immunoglobulin superfamily, is upregulated in ECs in response to inflammation and promotes leukocyte tethering through integrin receptor a4β1 (VLA-4) [21, 22], thereby stimulating progression of inflammation. Using VLA-4 inhibitor to block VCAM1-VLA-4 interaction, cortical neuron damage was ameliorated at 14d post 2VO. We concluded the continuous inflammatory response within blood vessels maybe an important factor in the continuous loss of cortical neurons following CCH.

MATERIAL AND METHODS

Animals

Adult male Sprague Dawley rats (body weight 280–300 g, 8–12 weeks old) were selected for this study and subjected to a 12-h light/dark cycle in a closed environment (24–26°C) with free access to food and water. Thirty rats were examined via immunohistochemistry, western blot (WB), and RT-PCR to observe the loss of cortical neurons, apoptosis, and the expression of inflammatory factors at different time points following CCH. Brain microvessels were extracted at various time points from fifteen rats in the sham group as well as in the CCH group for transcriptome sequencing. Thirty rats were injected with Evans blue (EB) dye through the tail vein to allow assessment of BBB permeability and detection of cerebral edema. The protective effect of injection with VLA-4 inhibitor on vascular inflammation was observed in 20 rats. All experimental procedures were approved by the Experimental Animal Committee of Zhengzhou University and the Henan Provincial People’s Hospital Committee and were performed in accordance with their standards.

Establishment of the CCH model

2VO operation-induced CCH was achieved. In short, surgery was performed under sterile conditions. Rats were positioned with abdomens facing up and were intraperitoneally injected with ketamine (50 mg/kg) and xylazine (10 mg/kg). A 2 cm midline incision was made on the ventral side of the neck of each rat. After careful separation of muscle tissue, nerves, and other adjacent tissues, bilateral common carotid arteries were identified and permanently ligated with filigree ligation. In sham surgery, the common carotid artery was exposed in the same way, but not ligated. Then, the muscle tissue and skin were sutured in layers. Finally, postoperative rats were placed on a thermal blanket for recovery.

Histology and immunohistochemistry

The rats in the sham group as well as rats from the 3d, 7d, 14d and 28d post 2VO groups were anesthetized and perfused with 100 ml normal saline and 500 ml 4% paraformaldehyde phosphate buffered fixative (PFA, pH 7.4) through the heart. Brains were then removed, fixed overnight in PFA, and finally cryoprotected in sucrose solution (30%) for 3–5 days. Cryosections (30μm) were prepared with a cryostat (Leica) and histologically examined. The primary antibodies used were as follows: rabbit anti-neuron (1:500, ab177487, 4587\yen, abcam); mouse anti-VCAM-1 (1:200, ab134047, abcam); mouse anti-IgG (1:500, 315-005-003, Jackson ImmunoResearch); Anti-Fibrin (1:500, ab34269, Abcam); Lectin (1:300, FL-1171, Vector Laboratories). HRP goat anti-rabbit IgG (1:100, A0208, Beyotime) and Alexa Fluor 488- conjugated goat anti-rabbit and 594-conjugated goat anti-mouse secondary antibodies (1:500, A-11078, A-11032, Thermo Fisher) were also used. Sections were examined using a BX53 microscope (Olympus).

Treatment with VLA-4 inhibitor

VCAM1 is a member of the immunoglobulin superfamily and is expressed on ECs at sites of inflammation. VCAM1 can interact with VLA-4 to help the recruitment of leukocytes to sites of inflammation which can promote the development of inflammation [23]. VLA-4 inhibitor can block VCAM1-VLA-4 interaction which can inhibit the further development of inflammation. 500μg of VLA-4 inhibitor (1.5 m g/kg, MCE) was injected through the tail vein of rats 4 h before and every day after 2VO surgery.

Measurement of brain water content and BBB permeability

Brain water content and BBB permeability were measured in the sham group and 3d, 7d, 14d and 28d postoperative groups. EB extravasation was used to evaluate BBB permeability. 2% EB (3 ml/kg, Sigma) was injected via tail vein at various time points. After 2 h of EB circulation, the rats were anesthetized and perfused with normal saline through the heart. The entire brain was collected and divided into left and right hemispheres. Brain water content was measured using the left hemisphere, while the right hemisphere was used to assess EB extravasation. To determine brain water content, the left hemisphere was weighed before and after dehydration in a 100°C oven for 24 h. Brain water content was quantified using wet/dry brain weight ratio which was used for statistical analysis. For the EB extravasation assessment, right hemisphere slices were weighed, homogenized with 1 ml of 50% trichloroacetic acid, and centrifuged at 10,000 rpm for 30 min. The supernatant was collected and mixed with an equal volume of ethanol. The concentration of EB was determined spectrophotometrically for absorbance at 620 nm. BBB permeability was evaluated by calculating the EB content (μg/g) according to the standard curve.

Extraction of rat brain microvessels

The rats in the sham group and 3d, 7d, 14d and 28d post operation groups were anesthetized and perfused with 100 ml of normal saline through the heart. Then, the brain was removed, the cortices were separated, and each cortex was placed in a petri dish containing pre-cooled DMEM high-glucose (HG) medium. Next, the tissue was cut, and 9 ml DMEM HG medium, 1 ml type II collagenase (final concentration is 1 g/L), and 100μl DNase I (15μg/ml) was added. The tissue was then digested at 37°C in a shaker (200 r/min) for 30 min. 5 ml DMEM HG medium was added to the mixture, which was then centrifuged at 1000 g for 8 min at 4°C. The supernatant was aspirated, and after adding 1 ml DMEM HG medium to resuspend, the mixture was transferred to a new 50 ml centrifuge tube. 12.5 ml 20% BSA-DMEM HG medium was added to resuspend, and the sample was centrifuged again at 1000 g for 8 min at 4°C. After aspirating the supernatant again, a red-white pellet (including abundant endothelial cells, a small number of astrocytes and pericytes) of microvascular tissue appeared at the bottom of the tube. 1 ml DMEM HG medium was added to resuspend the pellet and the final suspension containing cerebral microvessel fragments was transferred to a 1.5 ml sterile Ep tube.

Western blotting

Cortical tissue and brain microvessel fragments extracted from the cortex were digested in RIPA lysis buffer. Protein concentrations were quantified. Proteins were then separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes (Invitrogen, USA). After three washes in 0.05% Tween-20 (TBST), the membranes were blocked in 5% skim milk for 1 h at room temperature. Nitrocellulose membranes were incubated overnight at 4°C with mouse anti-VCAM1 (1:2000, ab134047, abcam), rabbit anti-il-1β (1:500, A1112, Proteintech), and rabbit anti-TNF-α (1:600, 17590-1-AP, Proteintech) primary antibodies. Then, membranes were incubated again with HRP-conjugated secondary antibody (1:1500, A0208, Beyotime) for 1 h at room temperature. Bands were visualized with Western Bright ECL solution and analyzed with Gel-Pro Analyzer 6.0 software.

RNA extraction

Cortical microvessel fragments extracted from the sham group and the 3d, 7d, 14d and 28d post 2VO groups were collected in TRIzol for RNA sequencing. Brain microvessel fragments from 3 rats were collected as a biological sample. Three biological replicates were performed for the sham group and the 3d, 7d, 14d and 28d post 2VO groups, respectively.

RNA sequencing and data analysis

Total RNA was extracted from microvessels to perform RNA sequencing analysis. The products were sequenced using Illumina NovaSeq 6000 by Gene Denovo Biotechnology Co. (Guangzhou, China). Raw reads from RNA sequencing were mapped to the reference genome using HISAT2 [24] (Version 2.1.0). Differentially expressed genes were identified using DESeq2 [25] based on the criteria that p-value < 0.05 and |log2Fold Change|>1.

RT-PCR

Reagents for reverse transcription and RT-PCR were purchased from Novizan. The primer sequences were as follows: VCAM1 primer: 5’- ACTGTGACCTGTCAGCGAAG-3’, 3’-TTAGGGACCGTGCAGTTGAC-5’; Il-1β primer: 5’-TTGAGTCTGCACAGTTCCCC-3’, 3’-GTCCTGGGGAAGGCATTAGG-5’; TNF-α primer: 5’-TTTCATACCAGGAGAAAGTC-3’, 3’-ATGACTCCAAAGTAGACCTG-5’; PAI-1 primer: 5’-GCTGGTGAACGCCCTCTATT-3’, 3’-CCATCCGGAGTGGTGAACTC-5’; egr-1 primer: 5’-AACAACCCTACGAGCACCTG-3’, 3’-AAAGGGGTTCAGGCCACAAA-5’; CCL2 primer: 5’-TAGCATCCACGTGCTGTCTC-3’, 3’-CAGCCGACTCATTGGGATCA-5’; ICAM-1 primer:5’-TTCCCTGGAAGGCCTGTTTC-3’, 3’-GGGAAGTACCCTGTGAGGTG-5’; CXCL2 primer: 5’-CGACCCTACCAAGGGTTGAC-3’, 3’-AGGTACGATCCAGGCTTCCT-5’; GADPH primer: 5’-TGCCACTCAGAAGACTGTGG-3’, 3’-TTCAGCTCTGGGATGACCTT-5’.

Cell counts

To quantify the number of neurons in the cortex, a randomly selected area from the slice was imaged using a 10x and 20x objective lens. Every cell expressing the selected marker was manually counted using Image-Pro Plus 7 (Media Cybernetics, USA). The data is presented as the average cell number in each field of view.

Statistical analysis

Multiple comparisons among groups were made by one-way ANOVA, followed by Dunnett’s post hoc test or two-way ANOVA. Data normality was assessed using the Shapiro– Wilk test. The data are presented as the mean±SD, and the boxplots show the maximum and minimum values. Statistical analysis was performed and graphs were made using GraphPad Prism 8.0 software. Values were considered significant at p < 0.05.

RESULTS

Persistent loss of cortical neurons after CCH

We began by evaluating cortical neuron loss at 5 time points in the sham group and the 2VO operation group at 3d, 7d, 14d and 28d after surgery. The results of immunofluorescence showed that, compared to the sham group, there was no significant change in cortical neurons at 3d post 2VO, while the number of cortical neurons at 7d, 14d and 28d post 2VO gradually decreased (Fig. 1A, C). This finding indicates that cortical neurons after CCH were continually lost. We further explored the mechanism of neuronal injury, harvesting cortical tissues at 5 time points to detect the corresponding apoptosis. The results of WB showed that, compared to the sham group, obvious apoptosis could be detected at 7d, 14d and 28d post CCH. Expression of the apoptosis-related proteins cleaved caspase-3 (Cas3) and Bax was increased (Fig. 1B, D, E). Our results suggest that, consistent with the loss of cortical neurons, cortical neuronal death following CCH may occur in the form of apoptosis. A previous study showed that, compared to the sham group, MAP2 immunofluorescence staining in cerebral cortical neurons was decreased and cortical Cas3 expression was increased in the mouse model 30 days after bilateral common carotid artery stenosis, which is consistent with our findings [11].

Fig. 1

Loss of cortical neurons following CCH. A, above) Pattern diagram of the frozen section sampling site. A, below) Pictures of immunofluorescence staining of cortical neurons in sham group and groups of rats 3d, 7d, 14d and 28d post CCH. B) WB analysis of the expression of Bax and cas3 in cortical tissue at various time points. C) Quantification of surviving cortical neurons at various time points. D,E) Quantification of Bax and cas3 expression in cortical tissue at various time points. n = 3 per group; NS, not significant; *p < 0.05, **p < 0.01, ***p < 0.001 compared to the Sham group; one-way ANOVA followed by Dunnett’s post hoc test.

BBB injury is an acute, transient process

The BBB precisely controls material exchange between blood and brain tissue, which is crucial for maintaining the stability of the brain microenvironment [26]. The dynamics of BBB integrity and the cascades that occur after CCH remain largely unclear. We examined changes in cortical BBB permeability following CCH. Increased brain water content indicates increased BBB permeability. We found that cerebral edema was the most severe 3 days after the operation and began to normalize 7d after the operation. Ultimately, brain water content returned to nearly normal levels after 2VO operation (Wet/dry weight ratio: 4.430±0.1718, 5.380±0.1351, 4.678±0.1242, 4.610±0.1597, 4.602±0.1152 in the sham, 3d, 7d, 14d and 28d groups, respectively) (Fig. 2A-C). Measurement of leakage of EB dye injected via the tail vein was also used to assess the integrity of the BBB. Upon gross examination of the brain, extravasation of EB was most obvious 3 days after the operation. The level of extravasation began to recover at 7d, and basically returned to normal by 14d. Changes in EB accumulation are consistent with changes in brain water content (EB concentrations: 2.068±0.6246, 7.844±0.2956, 2.910±0.3653, 2.458±0.6281, 2.1520±0.4293μg/g in the sham, 3d, 7d, 14d and 28d groups, respectively) (Fig. 2B, D). Previous findings also suggest that severe global barrier leakage occurs as early as 1 day post CCH, peaks at 3 days, and spontaneously recovers at 7 days after surgery [10].

Fig. 2

BBB injury following CCH. A) Schematic diagram of detection of BBB permeability. B) Gross anatomical changes in the brain in sham group and groups 3d, 7d, 14d and 28d after injection of EB. C) Time-dependent changes in post-operative brain dry-wet weight ratio. D) Quantification of EB staining at various time points. E) Brightfield staining of IgG leakage at various time points. F) Fluorescence staining of fibrin leakage at various time points. G,H) Quantification of IgG, fibrin staining at various time points. n = 5 per group; NS, not significant; **p < 0.01, ***p < 0.001 compared to the Sham group; one-way ANOVA followed by Dunnett’s post hoc test.

In addition, after the BBB breaks down, endogenous circulating macromolecules toxic to neurons infiltrate the brain (Fig. 2E, F). We observed IgG and fibrin deposition and leakage in the cortex via immunostaining. IgG leakage reached the most serious level 3d post operation, began to recover at 7d, and basically returned to normal by 14d (The IgG IOD: 26.62±5.888, 57.11±9.262, 41.19±2.738, 35.47±8.329, and 28.13±5.765, in the sham, 3d, 7d, 14d and 28d groups, respectively) (Fig. 2G), which is consistent with previous research findings [10]. Fibrin leakage reached the most serious level 3d post operation, began to recover at 7d, and basically returned to normal by 14d (The fibrin IOD: 30.58±6.729, 86.06±13.15, 50.52±11.81, 40.02±12.68, and 36.05±10.05 in the sham, 3d, 7d, 14d and 28d groups, respectively) (Fig. 2H).

The above results show that overall barrier damage was most serious 3d after CCH, began to recover at 7d, and basically returned to normal levels by 14d, and the accumulation of harmful substances (i.e., IgG, fibrin) following leakage did not persist in the brain. This suggests that damage to the BBB after CCH is an acute and transient pathological change, while the loss of neurons after CCH is persistent, and we speculate that damage to the BBB may not be an important cause of persistent neuron loss.

Brain microvascular fragment transcriptome sequencing

We selected 5 time points (sham group, and 3d, 7d, 14d and 28d post 2VO groups, referred to as group A, group B, group C, group D, and group E, respectively), and isolated cerebral microvessel fragments from the cortex of rats in each group. Fragments were subjected to bulk RNA-Seq. Compared with the group A, we found differential gene expression in group B (780 up, 478 down), C (628 up, 372 down), D (645 up, 418 down), and E (153 up, 87 down) (Fig. 3A). Out of a total of 1,231 differential genes, only 32 genes (2.6%) shared common changes at different time points pre- and post-injury (Fig. 3B). This indicates different functional states of cortical blood vessels at different time points after injury. GO analysis also showed phenotypic differences at various time points, and the differential genes observed at 3d were mainly attributable to changes in regulation of multicellular organismal processes, positive regulation of biological processes, anatomical structure development, and developmental processes (Fig. 3C, D). At 7d, the differential genes mainly indicated changes in cell activation, regulation of multicellular organismal processes, and response to stimulus (Fig. 3E, H), while at 14d, differential genes were mainly associated with immune and inflammatory responses (Fig. 3F, I). At 28d, differential expression mainly manifested as positive regulation of biological and developmental processes (Fig. 3G, J). These results indicate that the state of microvessels differs at each time point after injury. Further analysis found that compared to the 14d group, there were very few differential genes at 28d. VENN analysis confirmed that 84.9% of differential genes at 28d were also reflected at 14d (Fig. 4A). This shows that damage patterns tend to be consistent from 14d to 28d after injury. Therefore, in order to find the mechanism of neuron loss, we focused on analyzing the change in damage trends at 3d, 7d and 14d post injury. We defined 3d post 2VO as the acute phase, 7d post 2VO as the subacute phase, and 14d post 2VO as the chronic phase.

Fig. 3

Transcriptome sequencing analysis of cerebral microvessel fragments at various time points I: A) Compared with sham group, the number of differential genes at 3d, 7d, 14d and 28d post 2VO, respectively. B) VENN diagram of differential genes at various time points. C,E,F,G) GO analysis of differential genes in 3d, 7d, 14d and 28d groups compared to sham group post 2VO, bubble chart of significant enrichment, respectively. D,H,I,J) GO analysis of differential genes in 3d, 7d, 14d and 28d groups compared to sham group post 2VO bar chart of significant enrichment, respectively. n = 3 per group.

Fig. 4

Transcriptome sequencing analysis of brain microvessel fragments at various time points II: A) VENN map of differential genes at 14d and 28d post 2VO. B,D) KEGG analysis of differential genes in group 14d. C,E & G,H) Compared to differential genes in 3d group, GO analysis and KEGG analysis of differential genes unique to 14d group, modeled by significant difference bar graph and bubble chart, respectively. F, VENN diagram of differential genes in 3d and 14d group. n = 3 per group.

Cortical blood vessels are in a state of persistent inflammation

Previous studies have shown that BBB injury in the acute phase plays an important role in mediating acute brain parenchymal injury. Protecting the BBB in the acute phase can reduce the leakage of harmful substances, thereby reducing acute brain parenchyma injury [10]. Our results also suggest that BBB injury is most severe in the acute phase, begins to improve in the subacute phase, and has basically recovered by the chronic phase. However, the neuronal loss persists into the chronic phase, and there is no trend of improvement concurrent with recovery of the BBB (Figs. 1A and 2). Therefore, the functional status of cortical blood vessels at different periods following CCH is heterogeneous. Functional states during the acute and chronic phases are different, and the causes and mechanisms of neuronal loss are also different. To identify the cause of neuronal loss with respect to cortical blood vessels in the chronic phase, we further analyzed the functional status of cortical blood vessels at 14d (group D). We performed KEGG analysis on the differential genes in group D and found that MAPK signaling and cytokine-cytokine receptor interaction pathways were significantly changed (Fig. 4B, D). These pathological changes are closely related to vascular inflammation.

To clarify the specific changes within group D, we performed VENN analysis on the differential genes in group B and group D (Fig. 4F), and found that 168 genes were commonly changed, 530 genes were specifically changed in group B, and 304 genes were specifically changed in group D. These findings indicated that the genes that were differentially expressed in group B versus D were very different functionally. We selected 304 genes specifically altered in group D for GO analysis and KEGG analysis. GO findings revealed enrichment in inflammation-related functions (Fig. 4C, E). KEGG analysis found that cell adhesion molecules (CAMs), cytokine-cytokine receptor interaction, and other pathways were significantly changed (Fig. 4G, H). We found significant changes in specific genes related to cytokines, adhesion molecules, and other proinflammatory molecules. This indicated that cortical blood vessels were in a state of chronic inflammation at 14d post 2VO. RT-PCR verified these findings, and the results showed that the expression of inflammatory response-related factors, such as VCAM1, IL-1β, TNF-a, CXCL2, CCL2, ICAM-1, and other inflammatory response-related factors, was higher in brain microvessels at 14d post 2VO than in the sham group (Fig. 5G, H). WB results showed that expression of VCAM1, IL-1β, and TNF-α in cerebral microvessels was significantly higher at 14 days post 2VO than in the sham group (Fig. 5A-D). Immunofluorescence showed that expression of VCAM1 (co-stained with lectin) (Fig. 5E, F) in the cerebral microvessels was higher at 14 days post 2VO than in the sham group.

Fig. 5

Increase in cortical microvascular inflammatory indices 14 days post CCH. A) WB analysis of expression of VCAM1, TNF-a, and IL-1β in cortical microvessels in sham group and 14d group. B-D) Quantitative analysis of expression of VCAM1, TNF-α, and IL-1β in cortical microvessels in sham group and 14d group. E) Immunofluorescence analysis of VCAM1, lectin staining of cortical blood vessels in sham group and 14d group. F) Quantitative analysis of VCAM1, lectin staining in cortical blood vessels in sham group and 14d group. G,H) Quantitative RT-PCR analysis of VCAM1, IL-1β, TNF-a, ICAM-1, Egr-1, CXCL2, CCL2, and PAI-1 in cortical cerebral blood vessels in sham group and 14d group. n = 3 per group; NS, not significant; *p < 0.05, **p < 0.01, ****p < 0.0001 compared to the Sham group; one-way ANOVA followed by Dunnett’s post hoc test.

Blockade of the VCAM1 ligand VLA-4 reduces chronic inflammatory responses

VCAM1, a member of the immunoglobulin superfamily, is upregulated in endothelial cells in response to inflammation and promotes leukocyte tethering through VLA-4 [21, 22], thereby stimulating progression of inflammation. The pattern diagram is shown in Fig. 6A. Previous studies have shown that blocking VCAM1– VLA-4 interaction reduces seizures in a mouse model [27]. Therefore, we assessed the effect of VLA-4 inhibitor treatment on chronic inflammatory responses as well as modulation of cortical neuron loss status following CCH. 500μg of VLA-4 inhibitor was injected via tail vein 4 h before and every day after 2VO. We evaluated the loss of cortical neurons after VLA-4 inhibitor treatment. The results of immunofluorescence indicated that loss of cortical neurons was significantly alleviated after VLA-4 inhibitor treatment (Fig. 6B, C). This finding shows that VLA-4 inhibitor can effectively reduce the chronic inflammatory response of brain microvessels resulting from CCH, and consequently can improve neuronal loss. It confirms that the persistent loss of neurons in the cortex following CCH is most likely caused by the chronic inflammatory response in vessels.

Fig. 6

VLA-4 inhibitor can improve cortical neuron damage. A) Schematic representation of binding of VCAM1 to VLA-4 on endothelial cells. B) Quantitative analysis of survival of cortical neurons in the sham group, 14d group, and 14d group treated with VLA-4 inhibitor. C) Bright field staining of cortical neurons in sham group, 14d group, and 14d group treated with VLA-4 inhibitor. Inh., inhibitor. n = 4 per group; NS, not significant; *p < 0.05, **p < 0.01 compared to the Sham group. Unpaired t-test was to used.

DISCUSSION

CCH, a chronic state of decreased cerebral blood flow, is a common pathological process associated with many cerebrovascular diseases, including VaD, Alzheimer’s disease, atherosclerosis, carotid artery stenosis/occlusion, Moyamoya disease, and cerebral small vessel disease [28 –32]. CCH is a significant vascular risk that can lead to neuronal apoptosis, cognitive impairment, and neuroinflammatory responses [33 –35]. Neuronal apoptosis in particular plays a crucial role in the progression of CCH-induced cognitive impairment [36]. Cerebral vessels are responsible for delivering to the brain many important substances, such as nutrients and oxygen, which are required for neuronal oxidative metabolism of energy substrates. Neurons have only a limited capacity for anaerobic metabolism, so adequate cerebral blood flow is critical for neuronal function and viability [37]. Decreased cerebral perfusion reportedly correlates with dementia severity and predicts which patients with mild cognitive impairment will develop severe dementia [38, 39]. Therefore, preventing neuronal apoptosis may be an effective therapeutic strategy for treating CCH.

We used the 2VO rat model to simulate CCH. Supplementary Figure 1 shows animal numbers for various experiments and timepoints. We began by quantifying neuronal loss in the cerebral cortex of rats in the sham group and rats 3d, 7d, 14d and 28d post 2VO. Consistent with previous findings, we observed loss of neurons following CCH [10]. Moreover, the loss of cortical neurons continued to increase as time progressed, and there was no sign of slowing until 28 days after surgery. At the same time, results of WB of cortical tissue indicated that apoptosis within the cortex continued over time. It has been suggested that the cortex following CCH is in a state of continuous neuronal loss, which may occur in the form of apoptosis. CCH is considered to be a cause of BBB dysfunction [18]. By limiting the free diffusion of circulating toxins and pathogens, the BBB maintains a homeostatic brain microenvironment for healthy neural function [7, 8]. After the BBB breaks down, endogenous circulating macromolecules toxic to neurons infiltrate the brain, thereby damaging neurons and affecting cognition [40, 41]. Our previous study showed that severe global barrier leakage occurred 1 day post CCH, and it continued to worsen, reaching the worst level 3d after surgery, but spontaneous recovery was observed 7d after surgery [10]. Consistent with the results of previous studies, our study showed that BBB damage reached the most severe state at 3d post CCH, as evidenced by peak severity of EB dye extravasation, toxic protein leakage, and cerebral edema. BBB damage began to recover at 7d post 2VO in this study. Leakage of EB and toxic protein as well as cerebral edema were basically returned to baseline from 14d post 2VO. These results suggest that BBB injury after CCH is an acute, transient process.

The above results show that loss of cortical neurons following CCH is a continuous process, and there is no obvious change in this process with the recovery of the BBB. We speculate that damage to the BBB following CCH may not be the main reason for the continuous loss of neurons. To determine the reasons for continuous neuronal loss with regard to cortical blood vessels, we extracted cortical brain microvessel fragments at 5 time points for transcriptome sequencing. After analysis, it was found that the difference in the number of differential genes between the 14d and 28d groups was very small, indicating that the main changes observed at 28d had already existed at 14d. Knowing this, we designated the acute phase of injury as 3d post 2VO, the subacute phase as 7d, and the chronic phase as 14d after CCH. After GO and KEGG analysis, it was found that the main biological changes occurring in each phase were different, indicating that the functional states of cortical microvessels were different at various time points. Previous studies have mainly focused on the acute phase of BBB injury [10], studying the damage to the brain parenchyma in this state. Our findings showed that cortical neuron loss is a chronic state, so we shifted our focus to changes in microvascular expression during the chronic phase. After GO and KEGG analysis of the differential genes among groups B and D, it was determined that differential genes specific to group D were mainly concentrated in inflammatory pathways. These results suggested that in the chronic phase, vascular inflammatory responses may predominate, leading to neuron loss. Our results showed elevated levels of inflammatory factors, further validating this finding.

Previous studies have shown that under CCH conditions, many problematic mechanisms in the brain are chronically activated, leading to accumulation of damage. These mechanisms include long-term energy imbalance, oxidative stress, inflammation, endoplasmic reticulum stress, and mitochondrial dysfunction. These mechanisms drive downstream structural changes, such as white matter hyperintensity, glial activation, and cell death activation, resulting in a range of clinical symptoms [42, 43]. Additionally, an environment of hypoxic ischemia not only induces anaerobic metabolism in the brain, but also stimulates the release of pro-inflammatory cytokines from glial cells and neurons, resulting in further neurotoxicity in the brain [44] and subsequent damage to the brain parenchyma. Under hypoxic conditions in vitro, inflammatory mediators released by human astrocytes can upregulate the genes encoding IL-1β and TNF-α in human cerebral vascular endothelial cells [45], which supports our conclusion in vitro. In conclusion, both in vitro and in vivo studies have shown that ischemia and hypoxia can stimulate the body to produce an inflammatory response that damages the brain parenchyma. Vascular cell adhesion molecule VCAM1, a protein that promotes vascular-immune cell interaction, is among the inflammatory mediators released in hypoxic conditions VCAM1 is induced by a variety of inflammatory signals, and its gene expression is activated through a redox-sensitive mechanism that involves activation of the transcription factor NF-kB [46, 47]. As part of the inflammatory response, leukocytes act on vascular endothelial cells via VCAM1-VLA4 to maintain inflammation in endothelial cells [21, 22]. Previous literature has also demonstrated that vascular inflammation is associated with elevated VCAM1 levels, which mediate vascular remodeling [48]. By blocking VCAM1-VLA4 with VLA4 inhibitor in this experiment, it was found that the loss of cortical neurons was significantly reduced at 14d. It was further demonstrated that neuronal loss in the chronic phase following CCH is mainly caused by the chronic inflammatory response within blood vessels. Controlling vascular inflammatory response during the chronic injury phase following CCH is an important measure to mitigate cortical neuron loss during the chronic phase.

CONCLUSION

Cortical neuron loss resulting from CCH is a continuous process, and BBB injury may not be the main reason for persistent loss of cortical neurons. The persistent inflammatory response within blood vessels mediated by VCAM1 may be an important cause of chronic neuronal loss following CCH. Controlling the inflammatory response in the chronic phase of CCH injury may be an important process for treatment of persistent loss of cortical neurons in the chronic phase after resolution of acute CCH.

Footnotes

ACKNOWLEDGMENTS

We thank Henan Provincial Key Laboratory of Kidney Disease and Immunology, Zhengzhou University People’s Hospital, Henan Provincial People’s Hospital for technical assistance and data processing.

FUNDING

This work was supported by National Natural Science Foundation of China (Grants 82171196, 81873727); Henan Province Medical Science and Technology Co-construction Project (LHGJ20190779).

CONFLICT OF INTEREST

The authors have no conflict of interest to report.

DATA AVAILABILITY

Raw data of RNA sequencing are available through NCBI Sequence Read Archive database (SAMN32308253), at the following link: . All other data generated or used during the study appear in the submitted article.

The supplementary material is available in the electronic version of this article: .

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