Data of MSCs combined with LITUS treatment to improve cognitive impairment in a moderate traumatic brain injury model in rats

Here, we treated moderately traumatic brain injury (TBI) rats with different modalities, including transplantation with mesenchymal stem cells (MSCs), treatment with low-intensity transcranial ultrasound stimulation (LITUS), and a combination of the two. After the TBI rat model was established, MSCs (in situ injection within 24 h after injury), LITUS (continuous uninterrupted treatment for 28 days) or combined MSCs + LITUS were administered, and mNSS score, performance of behavior and multiple protein levels were compared between groups by behavioral observation, neurological function assessment and pathological analysis. Nestin, neuron-specific enolase (NSE), growth-associated protein 43 (GAP-43) and postsynaptic density protein (PSD-95) were significantly increased and glial fibrillary acidic protein (GFAP) was significantly decreased in the hippocampus of rats in the combination treatment group; brain-derived neurotrophic factor (BDNF), tumor necrosis factor-α (TNF-α) and aquaporin-4 (AQP-4) were significantly decreased in the injured peripheral cortex. The result of mNSS scores was: TBI group > LITUS group > MSCs group > MSCs+LITUS group > sham group. The alternate correct rate of Y-maze was: sham group > MSCs+LITUS group > MSCs group > LITUS group > TBI group. This data compares the efficacy of MSCs, LITUS, and combination therapy on the level expression of stem cell differentiation related proteins, synaptic plasticity-related proteins, neurotrophic factors, inflammatory factors, and edema-related proteins after TBI by quantitative pathological examination. For a complete description, interpretation, and discussion of the data refer to the article in press [1].

aquaporin-4 (AQP-4) were significantly decreased in the injured peripheral cortex. The result of mNSS scores was: TBI group > LITUS group > MSCs group > MSCs + LITUS group > sham group. The alternate correct rate of Y-maze was: sham group > MSCs + LITUS group > MSCs group > LITUS group > TBI group. This data compares the efficacy of MSCs, LITUS, and combination therapy on the level expression of stem cell differentiation related proteins, synaptic plasticityrelated proteins, neurotrophic factors, inflammatory factors, and edema-related proteins after TBI by quantitative pathological examination. For a complete description, interpretation, and discussion of the data refer to the article in press [1] .  Table   Subject  Biology  Specific subject area  Cognitive neuroscience and neural regeneration  Type of data  Tables, figures  How data were  acquired Rat neurological function scores were rated double-blind by two investigators according to the mNSS scoring criteria. The spontaneous alternation rate of the rats was calculated by the formula: correct alternation rate = number of correct alternations/(total number of arm entries -2) × 100%. The expression levels of relevant proteins and related genes in rat brain tissue were detected by immunohistochemistry, western blot and RT-PCR. Data

Value of the Data
• These data are useful because there is no effective treatment for TBI, which has high morbidity, mortality, and disability rates, and these data provide a reference for the treatment of TBI. • Clinicians and researchers in neurosurgery and rehabilitation, as well as the public, will benefit from this research, as the results have important implications for human health and disease. • These data can provide a reference for the treatment and rehabilitation after clinical TBI to improve the quality of life and survival of TBI patients. These data also • These data indicate the need for more research on stem cell combination therapy for TBI and encourage future studies to find more appropriate treatment parameters

Data Description
We applied MSCs, LITUS or MSCs + LITUS to rats with TBI and compared them with the control group without treatment as well as the sham group. The mNSS values of rats in each group on days 1, 3, 7, 14, 21, and 28 are shown in Table 1 , and the Y-maze spontaneous alternation rate is shown in Table 2 . Nesin, NSE, and GFAP protein expression in the DG area of the hippocampus; GAP-43 and PSD-95 protein and mRNA expression in the CA1 area; and BDNF, TNF-α, and AQP4 protein and mRNA expression values in the injured perirhinal cortex were detected by immunohistochemistry, western blot and RT-PCR in each group of rats, as shown in Tables 3-6 . Table 1 The mNSS values in each group of rats at 1, 3, 7, 14, 21 and 28 days after TBI (n = 9, mean ±SD). Data are presented as the mean ±SD. Values are compared with the TBI group. Differences were analyzed by one-way analysis of variance.

Table 2
The spontaneous alternation rate on the Y-maze for each group of rats 28 days after TBI (n = 6, mean ±SD). Data are presented as the mean ± SD. Values are compared with the TBI group. Differences were analyzed by one-way analysis of variance. * * p < 0.01. * * * p < 0.001. Data are presented as the mean ± SD. Values are compared with the TBI group. Differences were analyzed by one-way analysis of variance. * p < 0.05. * * p < 0.01. * * * p < 0.001.

Table 4
The mean optical density (AOD) values of GAP-43 and PSD95 in the hippocampal CA1 region of each group of rats 28 days after TBI (n = 6, mean ±SD). Data are presented as the mean ± SD. Values are compared with the TBI group. Differences were analyzed by one-way analysis of variance. * p < 0.05. * * p < 0.01. * * * p < 0.001.

Table 5
Relative protein and mRNA expression levels of GAP-43 and PSD95 in the hippocampal CA1 region of each group of rats 28 days after TBI (n = 6, mean ±SD).

GAP-43 PSD-95
Ratio Data are presented as the mean ± SD. Values are compared with the TBI group. Differences were analyzed by one-way analysis of variance. * p < 0.05. * * p < 0.01. * * * p < 0.001.

Table 6
Relative protein and mRNA expression levels of BDNF, TNF-α and AQP4 in the injured perirhinal cortex of rats in each group 28 days after TBI (n = 6, mean ± SD).

Rats and Groups
Ninety rats were randomly divided into five groups: 1 control group; 2 TBI group; 3 TBI + MSCs group; 4 TBI + LITUS group; 5 TBI + MSCs + LITUS group. A rat TBI model was established by controlled cortical impact (CCI) method using a pneumatic cranial precision percussion instrument (68099II, RWD, Shenzhen, Guangdong, China) [2] with the following relevant parameters: according to the rat brain stereotaxic map, a 5 mm diameter bone flap was grinding and drilling in the right parietal lobe (2.5 mm right of the sagittal line and 3.8 mm posterior to the coronal line of fontanelle), and the impact parameters were set as follows: the impact head diameter was 5 mm, the impact velocity was 5 m/s, the depth was 2.5 mm subdural, and the duration of impact was 1 s. MSCs were transplanted in situ at the central site of injury within 24 h after injury, and the relevant parameters were as follows: the cell suspension concentration was 2.5 × 10 7 cells/ml, the injection volume was 2 μL, the depth was 2.5 mm, the speed was 1 μL/min, and the needle retention time was 5 min [3] . LITUS treatment was performed daily without interruption for 28 days using an ultrasound stimulator (DK-102T, Dukang, Hebei, Shijiazhuang, China) with the following parameters: ultrasound frequency of 800 kHz, pulse duration of 10 ms, and pulse repetition period of 60 ms. The Isppa value was 1.2 W/cm 2 .

Behavioral Tests
Nine rats were randomly selected from each group, and mNSS scores were performed on days 1, 3, 7, 14, 21, and 28 after modeling [4] : the range of scores was 0-18, with higher scores representing more severe craniocerebral injury. Six rats were randomly selected from each group and the Y-maze test was performed on the 28th day after modeling: the Y-maze consisted of three 30 × 8 × 15 cm arms placed at a 120 °angle. Rats were recorded for 8 min after 2 min of free exploration in the maze, and entering three different arms consecutively was considered as one correct alternation. The alternation index is calculated as: correct alternation rate = number of correct alternations/(total number of arm feeds -2) × 100%.

HE Staining and Immunohistochemistry
After 28 days of modeling, six rats were randomly selected from each group, anesthetized by intraperitoneal injection of 2% sodium pentobarbital (2 ml/kg), and 4% paraformaldehyde was perfused to extract the brain for HE staining and immunohistochemical staining, as in the previous experiments [5] . The antibodies used for immunohistochemistry were as follows: rabbit monoclonal anti-Nestin

Western Blotting
The remaining rats were sacrificed and the brains were removed, the hippocampal tissue and the peri-injured cortex were isolated, and 100 mg samples were taken to detect the amount of protein by immunoblotting [6] . The antibodies used were as follows: rabbit monoclonal anti ies were detected using an ECL western blotting detection system kit (abs920, Absin Biotechnology Co., Ltd., Shanghai, China) and exposed using a ChemiDOC TM XRS + system with Image Lab TM software (Bio-Rad Life Medical Products Co., Ltd., Shanghai, China).

Semiquantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
A 100 mg sample was taken, total RNA was extracted from the tissue according to the manufacturer's instructions, amplified by a heat denaturation-retroactivity-extension cycle [7] , and PCR products were electrophoresed on a 1.5% agarose gel and visualized using a gel imaging system. The relative expression of each mRNA was calculated using the 2 − Ct relative quantification method.

Statistical Analysis
Data were analyzed by one-way ANOVA(one-way analysis of variance). Relative values of mNSS score, alternate correct rate, protein and mRNA were expressed as mean ± SD for each group of rats. Differences between groups were determined by LSD or Dunnett's test. SPSS software (version 22.0, Chicago, IL, USA) was used for statistical analysis of the data. p-value < 0.05 was considered a statistically significant difference.

Ethics Statement
The animal study was reviewed and approved by the Medical Ethics Committee of First Hospital of Qinhuangdao in China (ID Number: 20,140,018). All animal experimental procedures complied with the ARRIVE guidelines and were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability
Data of MSCs combined with LITUS treatment to improve cognitive impairment in a moderate traumatic brain injury model in rats (Original data) (Mendeley Data).