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Aggravated pulmonary injury after subarachnoid hemorrhage in PDGF-Bret/ret mice

Abstract

Background

Recent advances in surgical and neuroprotective strategies could effectively manage the pathophysiological progression of subarachnoid hemorrhage (SAH). However, pulmonary dysfunction frequently occurs in SAH patients with an increased risk of unsatisfactory outcomes. Based on the similar microvascular structures in the blood-air barrier and blood-brain barrier and possible brain-lung crosstalks, we believe that pericytes may be involved in both neurological and pulmonary dysfunction after SAH.

Methods

In our experiments, platelet-derived growth factor B (PDGF-B) retention motif knockout (PDGF-Bret/ret) mice and adeno-associated virus PDGF-B were employed to show the involvement of pericyte deficiency and PDGF-B expression. Neurological score, SAH grade, hematoxylin-eosin staining, and PaO2/FiO2 ratio analysis were performed to evaluate the neurological deficits and pulmonary functions in endovascular perforation SAH models at 24 h after surgery, as well as western blotting and immunofluorescence staining for underlying molecular expressions.

Results

We found that neonatal PDGF-Bret/ret mice exhibited pulmonary atelectasis 12 h after birth. Further investigation showed a decrease in PaO2/FiO2 and lung-specific surfactant proteins in adult PDGF-Bret/ret mice. These dysfunctions were much worse than those in wild-type mice at 24 h after SAH. PDGF-B overexpression alleviated pulmonary dysfunction after SAH.

Conclusions

These results suggested pulmonary dysfunction after SAH and the pivotal role of PDGF-B signaling for the pathophysiological process and future therapeutic targets of pulmonary injury treatment after SAH. Further studies are needed for pathophysiological investigations and translational studies on pulmonary injuries after SAH.

Background

Despite years of effort, subarachnoid hemorrhage (SAH) is still a complicated intracranial hemorrhagical stroke subtype associated with high morbidity and fatality, resulting in substantial loss to society. Recent advances in surgical and neuroprotective strategies could effectively manage the pathophysiological progression of SAH. However, pulmonary dysfunction frequently occurs in SAH patients with an increased risk of unsatisfactory outcomes [1], which was reported in approximately 10% of SAH survivors and almost half of deceased patients [2] without a proper therapeutic strategy. Neurogenic pulmonary edema is reported to be an important pathophysiological process that induces pulmonary dysfunction [3]. Based on the similar microvascular structures between blood-brain barrier and blood-air barrier and possible brain-lung crosstalks, we believe that pericytes may be involved in both neurological and pulmonary dysfunction after SAH and platelet-derived growth factor B (PDGF-B)-related signaling may not only control pericyte recruitment in cerebral microvasculature [4] but also act as an independent neurogenic mechanism for pulmonary dysfunction. Previous studies demonstrated that PDGF-B and PDGF receptor beta (PDGFR-β) were associated with the development of normal postnatal lung [5], as well as the incidence of cerebral vasospasm complication after SAH. So, we knockout and bred the PDGF-B retention motif in C57BL/6 genetic background mice (PDGF-Bret/ret mice) to investigate PDGF-B involvement in pulmonary injury after SAH.

Methods

As previously reported [6], we employed male PDGF-Bret/ret mice as knockout mice in our experiments, which were bred by the Shanghai Biomodel Organism Science and Technology Development Company Limited. Male Heterozygous PDGF-Bret/+ mice and wild-type PDGF-B+/+ mice were used as littermate controls. A total of 197 mice (4 weeks old, weighing 22 to 30 g) and 21 neonatals were employed in this study. All experimental procedures related to mice were approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University (no. AMUWEC2019449), complied with the guidelines by the National Institutes of Health 2011 Guide for the Care and Use of Laboratory Animals, and were presented in the format of the Animals in Research: Reporting In Vivo Experiments (ARRIVE) guidelines for purpose of replacement, refinement, and reduction of animals in research. Mice were bred in specific pathogen-free rooms of the Animal Center of Southwest Hospital, with controlled temperature and humidity 1 week before experiments and were given free access to water and food, along with half-day light/dark cycles.

Hematoxylin-eosin (HE) staining and western blotting were performed in neonatals at P0.5. Neurological score, SAH grade, hematoxylin-eosin staining, and PaO2/FiO2 ratio analysis were performed to evaluate the neurological deficits and pulmonary functions in endovascular perforation SAH models at 24 h after surgery, as well as western blotting and immunofluorescence staining for underlying molecular expressions. Details of these methods and statistical analysis are described as follows.

SAH model

As previously reported, endovascular perforation surgery was performed to induce SAH in mice [7]. All mice in the present study were intraperitoneally anesthetized with 1% sodium pentobarbital at the 40 mg/kg dosage. Briefly, start in the common carotid artery bifurcation, we slight migrated a sharpened 5–0 nylon suture into the distal internal carotid artery until slightly obstruct. After piercing the bifurcation between proximal middle cerebral artery and anterior cerebral artery, the resistance decreases. Then nylon suture was quickly extracted to restore blood flow and spread into intracranial subarachnoid space to mimic SAH onset. The mice in sham procedure had the same maneuver without perforating their vessels.

SAH grade assessment

Success of mice SAH models were evaluated by the grading the subarachnoid blood volume as previously described [8]. Briefly, according to the blood clot in subarachnoid, the basilar cistern was partitioned into 6 segments with a score from 0 to 3 for each segment. When calculating the total scores, add up the scores of all areas (maximum SAH score = 18). Due to the limited subarachnoid blood clot and mild neurological deficits, SAH mice with a score < 8 should be excluded from this study.

Adeno-associated virus administration

For in vivo adeno-associated virus (AAV) administration, the coding regions of pdgfb (738 bp) from C57BL/6J mouse cDNA were cloned into the AAV ITR-containing plasmid CMV-EGFP-2A-MCS-3FLAG, which became CMV-EGFP-2A-pdgfb. Recombinant AAV9 containing CMV-EGFP-2A-MCS-3FLAG (AAV9-EGFP) and CMV-EGFP-2A-pdgfb (AAV9-pdgfb) was manufactured in triple-transfection, helper-free procedures, and then purified by OBIO Technology (Shanghai) Corp., Ltd. A total of 75 μL sterile PBS solution of 1012 v.g. AAV9-pdgfb was administered by intratracheal intubation to each mouse 4 weeks before SAH to overexpress full-length PDGF-B protein in the lung, and AAV9-EGFP served as a control.

Hematoxylin and eosin staining

Hematoxylin and eosin staining were performed as described previously [9]. Lung specimens were removed and post-fixed for a minimum of 24 h in 4% paraformaldehyde solution, then paraffin embedded and slice into the 8-μm-thick sections on a vibratome machine. These sliced sections were infiltrated in xylene for deparaffinizing and then in decreasing gradient ethanol solutions for rehydration. Then, the sliced sections were placed in glass slides and stained with the hematoxylin dye and eosin dye for further observation.

PaO2/FiO2 ratio analysis

To evaluate pulmonary function after SAH, the PaO2/FiO2 (P/F) ratio was calculated as below. At 24 h after mimic SAH onset, mice were similarly intraperitoneally anesthetized with 1% sodium pentobarbital at the dosage of 40 mg/kg and endotracheally intubated with a 22-gauge catheter for mechanical ventilation with 21% oxygen. The respiratory frequency was 18 breaths per minute. Arterial blood sample was collected from the carotid artery and measured by following the operator’s manual in blood gas analyzer.

Western blot

Western blot analysis for protein expressions were performed as reported previously in our laboratory [7]. Lung specimens were collected after mice sacrifice and lysed in 20 mM Tris for following standard western blot analysis. Primary antibodies were list as follow: anti-SP-B antibody (Abcam, Cambridge, MA), anti-SP-C antibody (Abcam, Cambridge, MA). Primary α-tubulin antibody from Beyotime Biotechnology (Shanghai, China) was loaded as the internal control for normalization between different groups. The corresponding secondary antibodies were then incubated and imaged with an ECL+Chemiluminescent Kit (Amersham Bioscience, Arlington Heights, IL) to identify the immune bands. Further analysis was performed by densitometry quantified with Quantity One software (Bio-Rad, Berkeley, CA).

Immunofluorescence staining

The protein expression in lung specimens was also evaluated by immunofluorescence staining on fixed frozen sections similar to HE staining as previously described [7]. Brain sliced sections on coverslips were fixed in 4% paraformaldehyde. Samples were blocked by 5% donkey serum prior to incubation with primary antibody overnight with the following antibodies: anti–SP-C (Abcam, Cambridge, MA) at 4 °C overnight, followed by fluorescein-conjugated antibodies for immunofluorescence (Beyotime Biotechnology, Shanghai, China). Then, the sliced sections were placed on glass slides for laser-confocal microscope observation.

Statistical analysis

SPSS 18 (SPSS China, Shanghai, China) was used for the statistical analyze in the present study. Data are presented in the format of mean ± standard deviation (mean ± SD). Neurobehavioral scores were analyzed by using chi-square tests. One-way analysis of variance (ANOVA) plus Tukey’s multiple comparisons test was used for multiple comparisons of more than three groups. If the P < 0.05, the comparison among groups was considered significantly different.

Results

PDGF-B involvement in pulmonary injury in neonatal mice

Hematoxylin-eosin staining indicated that neonatal PDGF-Bret/ret mice exhibited pulmonary atelectasis at 12 h after birth (P0.5), which was less severe in PDGFret/+ mice (Fig. 1a). This phenomenon was associated with significantly lower SP-B and SP-C expressions in the pulmonary tissues of neonatal PDGF-Bret/ret mice at P0.5 (P < 0.05, Fig. 1b, c).

Fig. 1
figure1

Pulmonary injury in neonatal PDGFret/ret mice. a Representative HE staining images of the lung parenchyma in neonatal PDGFret/ret mice and controls at 12 h after birth. Representative bands and quantitative graphs of b SP-B and c SP-C expressions in pulmonary specimens at 12 h after birth. Relative densities were controlled with α-tubulin. Those western blot bands were collected under the same conditions and cropped form better exhibition effect. N = 6, @: vs. +/+ P < 0.05, #: vs. ret/+ P < 0.05. Scale bar 100 μm\

In addition, PDGF-B protein expression was significantly decreased at 24 to 72 h after mimic SAH in adult PDGF-B+/+ mice (P < 0.05, Fig. 2a, b). Immunofluorescence indicated that the pericytes in the lungs were greatly reduced in PDGF-Bret/ret mice at both the neonatal and adult stages compared to those in PDGF-B+/+ mice (Fig. 2c). These images also indicated that the pericyte deficits in the lung tissues of PDGF-Bret/ret neonatal mice were not normalized during adulthood (Fig. 2c).

Fig. 2
figure2

PDGF-B expression in the lungs after SAH and pericytes in wild-type and PDGFret/ret mice at both the neonatal and adult stages. a Immunoblot bands and b quantitative graphs of PDGF-B expression in lung specimens at 24 h after mimic SAH onset. Relative densities were controlled with α-tubulin. Those immunoblot bands were collected under the same conditions and cropped form better exhibition effect. c Representative immunofluorescence images of PDGFRβ (green) and Lectin (red) expressions in the lung parenchyma of wild-type PDGF+/+ and PDGFret/ret mice at both the neonatal and adult stages. Relative densities were controlled with α-tubulin. N = 6, @: vs. sham P < 0.05. WT: wild-type, ret: PDGFret/ret, Scale bar 50 μm

Pulmonary dysfunction after SAH in adult PDGF-Bret/ret mice

At adult stage, there is no mice died after sham-operated, and 13 mice died after SAH modeling, including 4 PDGF-B+/+ and 9 PDGF-Bret/ret mice. And the grading scores have no significant differences to confirm the consistence SAH model induced in our study (P > 0.05, Fig. 3a). The survival rates of PDGF-Bret/ret mice were much lower comparing to the PDGF-B+/+ mice at 48 or 72 h timepoints after mimic SAH onset (P < 0.05, Fig. 3b). Neurological deficits did not differ between PDGF-Bret/ret and PDGF-B+/+mice at the timepoint of 24 h after mimic SAH onset (P > 0.05, Fig. 3c, d). PaO2/FiO2, a key indicator of pulmonary function, in the wild-type group was reduced at 24, 48, and 72 h after SAH versus the sham-operated mice (P < 0.05, Fig. 4a), which demonstrated pulmonary injury existing post-SAH. Further investigation demonstrated that PaO2/FiO2 in PDGF-Bret/ret mice at 24 h after SAH was significantly lower comparing to the PaO2/FiO2 in PDGF-B+/+ mice (P < 0.05, Fig. 4b). These results indicated aggravated pulmonary injury after SAH in PDGF-Bret/ret mice.

Fig. 3
figure3

Survival percent and neurological functions after SAH. a The SAH grading of mice among groups at 24 h after SAH. b The survival percent at 12, 24, 48, and 72 h in each group. c, d Neurological evaluation scores of mice among groups at the timepoint of 24 h after mimic SAH onset. WT: wild-type, ret/ret: PDGFret/ret, PDGF-B: AAV9-pdgfb. N = 20 for percent survival analysis, N = 6 for others, @: vs. WT + sham P < 0.05, & in panel a: vs. WT + PDGF-B + sham P < 0.05, & in other panels: vs. WT + SAH P < 0.05, # in panel a: vs. ret/ret + sham P < 0.05, # in other panels: vs. WT + SAH P < 0.05

Fig. 4
figure4

Pulmonary dysfunction after SAH in adult PDGFret/ret mice. a The time course of the PaO2/FiO2 ratio after SAH in wild-type mice. b The PaO2/FiO2 ratio at the timepoint of 24 h after mimic SAH onset. c HE staining pictures of the lung parenchyma. WT: wild-type, ret/ret: PDGFret/ret, PDGF-B: AAV9-pdgfb, Scale bar 200 μm. N = 6, @: vs. WT + sham P < 0.05, # in panel b: vs. WT + sham P < 0.05, # in panel a: vs. sham P < 0.05

Pulmonary dysfunction after SAH associated with low lung-specific surfactant proteins

Pulmonary autopsy showed similar pulmonary atelectasis at 24 h after SAH in PDGFBret/ret mice, meanwhile, the PDGFB+/+ SAH mice were much better at the same time point (Fig. 4c). Western blotting showed that much lower SP-B and SP-C expressions in pulmonary tissue after SAH, and much lower expressions in PDGF-Bret/ret mice comparing with PDGF-B+/+ mice (P < 0.05, Fig. 5a, b). These results were confirmed with immunofluorescence staining, which showed less fluorescence intensity in PDGF-Bret/ret mice comparing with PDGF-B+/+ mice at 24 h after SAH (Fig. 5c).

Fig. 5
figure5

Expression of lung-specific surfactant proteins after SAH in adult PDGFret/ret mice. Immunoblot bands and quantitative graphs of a SP-B and b SP-C expressions in pulmonary specimens at 24 h after mimic SAH onset. Relative densities were controlled with α-tubulin. Those immunoblot bands were collected under the same conditions and cropped form better exhibition effect. c Representative immunofluorescence images of SP-C expression in lung parenchyma at 24 h after mimic SAH onset. WT: wild-type, ret/ret: PDGFret/ret, PDGF-B: AAV9-pdgfb, N = 6. @: vs. WT + sham P < 0.05, #: vs. WT + sham P < 0.05. Scale bar 10 μm

Pulmonary dysfunction after SAH was alleviated after PDGF-B overexpression

To be more translational, we employed adeno-associated virus to overexpress PDGF-B in adult wild-type mice. The results indicated that PaO2/FiO2 was significantly increased in PDGF-B-overexpressing mice at 24 h after SAH compared to normal PDGF-B+/+ SAH mice (P < 0.05, Fig. 4b), which demonstrated that pulmonary injury after SAH was alleviated by PDGF-B overexpression. This phenomenon was also connected with increased SP-B and SP-C expressions in pulmonary tissue (P < 0.05, Fig. 5a–c). Meanwhile, as previously reported to be neuroprotective [10], PDGF-B overexpression significantly improved the survival percent and neurological outcomes after SAH in adult wild-type PDGF-B+/+ mice (P < 0.05, Fig. 3b–d).

Discussion

In the present study, neonatal PDGF-Bret/ret mice exhibited pulmonary atelectasis at P0.5, which confirmed the involvement of PDGF-B in pulmonary injury. Further investigation showed a decrease in PaO2/FiO2 and lung-specific surfactant proteins and evidence of pulmonary dysfunction after SAH in adult PDGF-Bret/ret mice, which were much worse than wild-type mice at 24 h after SAH. PDGF-B overexpression alleviated pulmonary dysfunction after SAH. These results suggested pulmonary dysfunction after SAH and indicated the pivotal role of PDGF-B signaling for pathophysiological process and future therapeutic targets of pulmonary injury in SAH patients.

PDGFs serves as dimers of A, B, C, D single chains and ligands interact with its tyrosine-kinase alpha and beta receptors, short named as PDGFR-α and PDGFR-β. A previous study suggested that PDGF was induced in cerebrospinal fluid and vasculature, associated with an enhanced contractile response and vasospasm after SAH[11, 12]. PDGFA−/− mice developed secondary lung emphysema after alveolar septation failure, which may be the result of low elastin expression [13]. Overexpress PDGF-A exhibited fatal pulmonary atelectasis at embryonic day 18.5 and could not survive after birth, with increased macrophages/eosinophils in airspaces and reduced elastin expression and emphysema [14]. Our results demonstrated that PDGF-B signaling was involved in pulmonary injury, with physiological changes in pulmonary atelectasis and reduced lung-specific surfactant protein. Consistently, PDGF-B antisense would inhibit fetal lung cell growth [15], suggesting a possible protective role of PDGF-B signaling for the lung development and repair. After exposure to a high concentration (60%) of oxygen, PDGF-B mRNA, but not PDGF-A, was increased in the lung parenchyma compared to the air-exposed lung after birth [5], indicating that PDGF-B might be more sensitive to hypoxia in the brain and lung after SAH. However, the fact that PDGF-Bret/ret mice have lung defects at baseline from birth may somehow influence the evaluation of pulmonary dysfunction after SAH.

In our experiments, we knocked out retention motif of PDGF-B, which is the binding site of PDGF-B with the extracellular matrix [16]. Previous studies demonstrated that PDGF-Bret/ret mice suffered cerebral vascular dysfunction by pericyte recruitment deficiency and subsequent breach on the blood-brain barrier after SAH [417,18,19,20]. Similarly, the pericytes were covered with 26–40% microvessels in mature lungs [21], which might also be regulated by PDGF-B in the pathophysiological progression after SAH to cause pulmonary edema, previously considered to be neurogenic [22]. Based on our results, the PDGF-B signal played an important role in pulmonary injury after SAH, including pulmonary elastin expression, lung-specific surfactant proteins, and blood oxygen exchange. However, due to the limitations of the present study, we cannot exclude other possible mechanisms of pulmonary injury after SAH, such as FOXF1 maintaining endothelial barriers and preventing neurogenic edema after lung injury [23].

Conclusion

PDGF-B might serve as a potential therapeutic target for pulmonary dysfunction after SAH and greatly change the situation of merely supporting strategies for this severe peripheral complication in SAH patients. Further studies are still needed for pathophysiological investigations and translational studies on pulmonary injuries after SAH.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Abbreviations

SAH:

Subarachnoid hemorrhage

PDGF-A/B:

Platelet-derived growth factor A/B

PDGFR-α/β:

Platelet-derived growth factor receptor alpha/beta

PDGF-Bret/ret :

PDGF-B retention motif knockout

ARRIVE:

Animals in Research: Reporting In Vivo Experiments

AAV:

Adeno-associated virus

P/F:

PaO2/FiO2

SP-B/C:

Lung-specific surfactant protein B/C

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Acknowledgements

We sincerely appreciated the Shanghai Biomodel Organism Science and Technology Development Co., Ltd. for the technical support on breeding the PDGF-Bret/ret mice.

Funding

This study was supported by the Top-notch Talent Cultivation Plan of Southwest Hospital (SWH2018BJKJ-05), Natural Science Foundation of Liaoning Province (20180550504) and the Major Innovation Project of Southwest Hospital (SWH2016ZDCX1011).

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Contributions

Y.C. and H.F. conceived and designed the experiments; P.P., J.Q., Q.L., R.L., Y.Y., and S.Z. performed the experiments; P.P., X.L., and Y.C. analyzed the data; Y.C. and P.P. wrote the manuscript. Y.C. gave the final approval for the manuscript to be published. The manuscript has been read and approved by all the authors, that the requirements for authorship have been met, and that each author believes that the manuscript represents honest work.

Corresponding author

Correspondence to Yujie Chen.

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Ethics approval and consent to participate

All animal procedures were approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University (no. AMUWEC2019449), performed in accordance with the guidelines by the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and reported by following the Animals in Research: Reporting In Vivo Experiments (ARRIVE) guideline.

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The authors declare that they have no competing interests

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Pan, P., Qu, J., Li, Q. et al. Aggravated pulmonary injury after subarachnoid hemorrhage in PDGF-Bret/ret mice. Chin Neurosurg Jl 6, 13 (2020). https://doi.org/10.1186/s41016-020-00193-2

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Keywords

  • Platelet-derived growth factor B
  • Pulmonary injury
  • Subarachnoid hemorrhage
  • Lung-specific surfactant protein