A Novel Cranial Bone Transport Technique Repairs Skull Defect and Minimizes Brain Injury Outcome in Traumatic Brain Injury Rats

Abstract

TBI (traumatic brain injury) is a major cause of mortality and morbidity among young adults with limited therapeutic strategies. Cranial bone transport (CBT) technique is a safe, less invasive, and relatively simple surgical technique in bone reconstruction, which has been used to repair cranial bone defects and deformity corrections. The current studies are to determine the effects of CBT surgery on cranial bone regeneration as well as neurological functional recovery in TBI. CBT treatment alleviated lesion size and promoted learning, motor, and memory recovery in TBI rats. The meningeal lymphatic drainage function is enhanced, evidenced by increased intake of ovalbumin conjugated with Alexa Flour 647(OVA-A647) in meningeal lymphatic vessels (MLVs) and deep cervical lymph nodes (dCLNs). CBT accelerated P-tau clearance while decreasing Iba1 induced neuroinflammatory response in TBI rats. Notably, improvement of CBT treatment is significantly abolished by the ablation of MLVs via MAZ51, a small-molecule inhibitor primarily targeting vascular endothelial growth factor receptor-3 (VEGFR-3). Furthermore, after bone transport treatment, bone regeneration in the CBT sites continued consolidation, bone defects in TBI are replaced with new bone more quickly after CBT surgery. Taken together, the study is a proof-of-concept de-novo study to prove CBT can significantly improve the outcomes of brain recovery and cranioplasty in TBI rats.

Keywords: cranial bone transport; cranioplasty; meningeal lymphatic drainage; neuro‐inflammation; traumatic brain injury.

Figure 1

CBT reduced lesion size, attenuated neuronal death, and improved motor, memory, cognitive function in TBI rats. A) Schematic diagram of experiment design. B) Representative Nissl staining and images of gross view of four groups. C) Quantification of percentage of lesion size, calculated by ratio of the lesion area in ipsilateral brain to whole area of contralateral brain. D) Representative image of perilesional cortex section showing NeuN+ immunofluorescence at 14 days after TBI, scale bar = 100 µm. E) Quantification of total viable neurons in the perilesional cortex. F) Time to fall from the rotating rod of TBI rats significantly increased after 8 days of BT surgery. G) Representative images of track in spatial memory test part for four groups. H) Histograms represent the duration in B arm for each experimental group. I) Histograms represent the preference index for each experimental group. Preference index = time spent with novel object/total exploration time. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001. Data are shown as mean ± SD.

Figure 2

Augmentation of meningeal lymphatic drainage after CBT in TBI rats. A) Schematic of the experimental layout. Rats were injected intra‐cisterna magna (i.c.m.) with OVA647, then dCLNs and brains were collected after 2 h. B) Representative images of MLVs in middle meningeal artery region with OVA‐A647 (red) stained with DAPI (blue), scale bar = 100 µm. C) Representative images of dCLNs with OVA‐A647 (red) stained with DAPI (blue), scale bar = 500 µm. D) Quantification of OVA‐A647 in MLVs in four groups. E) Quantification of OVA‐A647 in dCLNs in four groups. F) Quantification of diameter of MLVs in the four groups. G) Quantification of MLVs coverage in four groups. H) Representative images of perilesional cortex stained with P‐tau (red) and DAPI (blue), scale bar = 50 µm. I) Quantification of P‐tau fraction in four groups. *p<0.05, **p<0.01, ***p<0.001. Data are shown as mean ± SD.

Figure 3

CBT alleviated TBI‐induced neuroinflammation and microglia activation. A) Representative images of the cortex region in TBI brain in the four groups, scale bar = 50 µm. B) Representative images of the hippocampus region in TBI rat brain in the four groups, scale bar = 50 µm. C‐D) Quantification of the percent area of GFAP (C) and Iba1 (D) immunoreactivity per field of view in the cortex by Image J software. (E‐F) Quantification of the percent area of GFAP E) and Iba1 F) immunoreactivity per field of view in the hippocampus by Image J software. G) Representative reconstructions of morphology of Iba1+ cells. H) Sholl analysis of Iba1+ cells interactions in four groups. (#) indicates that significant differences between TBI and TBI+BT groups. *p<0.05, **p<0.01, ***p<0.001. Data are shown as mean ± SD.

Figure 4

Ablation of meningeal lymphatic vessels reversed the therapeutic effects of CBT. A) Representative Nissl staining and images of gross view of the TBI rats injected with MAZ51. B). Quantification of percentage of lesion size, calculated by ratio of the lesion area in ipsilateral brain to whole area of contralateral brain. C) Time to fall from the rotating rod of TBI rats significantly increased after 3, 8, and 13 days of BT surgery. D) Representative images of Y maze track in three groups. E) Duration of rat staying in novel arms of Y maze in three groups. F) Histograms represent the discrimination index (DI) for each experimental group. Discrimination index = (time spent with novel object − time spent with old object)/total exploration time. G) Representative images of perilesional cortex stained with GFAP (green) and Iba1 (red), scale bar = 50 µm. H) Representative images of perilesional cortex stained with P‐tau, scale bar = 50 µm. I) Quantification of Iba1 fraction in three groups. J) Quantification of GFAP fraction in three groups. K) Quantification of P‐tau fraction in three groups. ^* p < 0.05, ^** p < 0.01, ^*** p <0.001. Data are shown as mean ± SD.

Figure 5

CBT enhanced bone healing and promoted osteogenic marker expression in TBI skull defects. A) Schematic of the experimental layout. CBT surgery was performed 24 h after the establishment of TBI model, after BT treatment for 13 days, and a bone consolidation period for further 2 months. Skull samples were collected for micro‐CT and histology analysis. B) Representative images of micro‐CT reconstruction in the two groups. Scale bar = 1 mm. C) Quantitative bone volume analysis (BV: bone volume; TV: total defect volume). D) Quantitative bone mineral density measurement. (E) Representative images of H&E staining in the two groups, scale bar = 100 µm. B: border of bone defect; NB: new bone, F: fibrous tissue. F) Representative images of Masson staining in the two groups, scale bar = 100 µm. G,I) Representative images of COL‐1 and OCN immunohistochemical staining of the skull, scale bar = 100 µm, H) Quantification of COL‐1 positive area of bone defects region in two groups. J) Quantification of OCN positive area of bone defects region in two groups. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001. Data are shown as mean ± SD.

Figure 6

CBT treatment reduced lesion size and increased the neuronal cells survival in the perilesional cortex 2 months post‐TBI. A) Representative Nissl staining and images of gross view of TBI rats treated with CBT after 2 months. B) Quantification of percentage of lesion size, calculated by ratio of the lesion area in ipsilateral brain to whole area of the contralateral brain. C) Quantification of total viable neurons in the perilesional cortex. D) Representative image of the perilesional cortex section showing NeuN+ immunofluorescence at 2 months after TBI, scale bar = 100 µm. E) Representative image of the striatum section showing NeuN+ immunofluorescence at 2 months after TBI, scale bar = 100 µm. F) Representative image of the hippocampus section showing NeuN+ immunofluorescence at 2 months after TBI, scale bar = 100 µm G) Quantification of total viable neurons in the striatum. H) Quantification of total viable neurons in the hippocampus. I) Representative images of perilesional cortex stained with P‐tau (red) and DAPI (blue), scale bar = 50 µm. J) Quantification of P‐tau fraction in two groups. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001. Data are shown as mean ± SD.

Figure 7

An 18‐year man suffered TBI was performed CBT surgery. (A‐B) Structure of the external fixator: anterior view A) and dorsal view B). C) Patient was in a state of mild disturbance of consciousness with a trachea intubation to maintain ventilation. D) CT reconstruction showed a bone defect in the left skull after decompressive craniectomy for about 4 months. E) Representative CT Image of horizontal position indicated large defects in parietal region. F) A bone flap was designed to be obtained in the rim of bone gap border. G) View of patient with the external fixator after surgery.

Figure 8

Therapeutic effects of CBT on case 1. A) Patient was able to feed himself 2 months after operation. B) He can walk slowly after removal of the external device. C) X‐ray results suggested bone transport repaired the bone gap. D) Representative CT Image of coronal position showed defects in temporal region were reduced. E) Representative CT Image of horizontal position showed bone defects were repaired.