TNF‐α interference ameliorates brain damage in neonatal hypoxic–ischemic encephalopathy rats by regulating the expression of NT‐3 and TRKC

Abstract The aim of this study is to explore the effect of tumor necrosis factor‐α (TNF‐α) inhibition in rats with neonatal hypoxic–ischemic encephalopathy (HIE) and ascertain the relevant signaling pathways. The Zea–Longa score was used to evaluate the neurological function of the rats. ImageJ was used for quantification of the brain edema volume. Triphenyl tetrazolium chloride (TTC) staining of brain tissue was performed 24 h after hypoxic–ischemic (HI) to detect right brain infarction. The expression of TNF‐α was detected by real‐time quantitative polymerase chain reaction (RT‐qPCR). Immunofluorescence staining was used to identify the localization of TNF‐α; Then, the effective shRNA fragment of TNF‐α was used to validate the role of TNF‐α in HIE rats, and the change of neurotrofin‐3 (NT‐3) and tyrosine kinase receptor‐C (TRKC) was examined after TNF‐α‐shRNA lentivirus transfection to determine downstream signaling associated with TNF‐α. Protein interaction analysis was carried out to predict the links among TNF‐α, NT‐3, and TRKC. Cerebral edema volume and infarction increased in the right brain after the HI operation. The Zea–Longa score significantly increased within 24 h after the HI operation. The relative expression of TNF‐α was upregulated after the HI operation. TNF‐α was highly expressed in the right hippocampus post HI through immunofluorescence staining. Bioinformatics analysis found a direct or an indirect link among TNF‐α, NT‐3, and TRKC. Moreover, the interference of TNF‐α increased the expression of NT‐3 and TRKC. TNF‐α interference might alleviate brain injury in HIE by upregulating NT‐3 and TRKC.

2][3] It increases the rate of death and lifelong disability due to epilepsy, learning disabilities, intellectual deficiency, and brain paralysis. 4The morbidity of HIE in developed and developing countries is 1.5 and 10-20 per 1000 live births, respectively. 5Worse still, brain damage occurs in five out of every 1000 live births. 6urrent medical support and hypothermia are standard treatments for moderate to severe HIE, which has been shown to reduce mortality, but 20% of infants with HIE still die. 7To date, there are few treatment options for HIE, and the huge financial burden for most families and reduced quality of life caused by HIE are major problems.Therefore, novel and effective treatments must be developed for effective treatment of HIE.
][10][11] TNF-α, as a cell signaling protein, is one of the cytokines that constitutes the acute phase of the reaction. 124][15] It has been demonstrated that full-term neonates with HIE have elevated TNF-α levels and TNFα can aggravate the degree of brain injury by reducing neuronal cell viability in the brain. 16,17TNF-α can disrupt the blood-brain barrier and damage cortical cells to aggravate brain injury. 18,19Moreover, it has been shown that TNF-α is a negative factor in HIE and 25 anti-TNF agents are used to treat inflammatory diseases with TNF dysregulation, but excluding HIE. 20n this study, the impact of TNF-α inhibition and its related molecular mechanism were further investigated, so as to find a new strategy for the treatment of HIE.This is helpful for the application of TNF-α inhibitors in HIE treatment.

| Establishment of neonatal rat HI models
Seven-day-old SD rats were weighed and numbered before administration of 3% isoflurane anesthesia.Before the operation, the hypoxia chamber was set up with a temperature of 37°C and humidity of 50%-80%.A 0.5 cm skin incision was made in the midline of the neck with scissors to expose the right common carotid artery (CCA), which was blocked by an electrocautery device (Spring Medical Beauty Equipment Co, Ltd).After recovering for 1 h beside their mothers, the postoperative rats were placed into the hypoxia chamber with 8% O 2 and 92% N 2 (flow velocity 3 L/min) for 2 h after waiting with their mothers for 1 h, then taken out and placed together with their mothers.For anesthetized rats in the sham group, only the right CCA at the skin incision was separated without right CCA ligation and oxygen deficiency.

| Zea-Longa score
Over six periods, pre-operation, and 0, 4, 8, 12, and 24 h after the HI operation, the Zea-Longa score was used to assess the severity of nerve injury and to determine the success of model construction, and the scoring criteria were quintile: 0, normal without nerve injury; 1, the forelimb on the contralateral side of the injury could not be extended; 2, tending to rotate on the contralateral side of the injury when crawling; 3, dumping on the contralateral side of the injury when crawling; and 4, could not walk and lost consciousness.

| Sample acquisition
Rats were anesthetized and the thoracic cavity was rapidly opened to expose the heart 24 h after surgery.Then, the tip of the 50-ml syringe was inserted into the ascending aorta from the apex and perfused with 0.9% saline.Afterwards, brain tissues were quickly removed and frozen at −20°C for 10 min for triphenyl tetrazolium chloride (TTC) staining.Fresh samples of the hippocampus, cortex, lung, and heart were directly placed in RNase free EP tubes and frozen at −80°C for real-time quantitative polymerase chain reaction (RT-qPCR).For immunofluorescence staining, brain tissues were isolated, fixed with 4% paraformaldehyde (4°C, pH = 7.4) at 4°C for 5 h, and stored in 0.1 Mol (M) phosphate buffer containing 30% sucrose at 4°C for 72 h.Finally, brain tissues were embedded in paraffin and serially cut into 5 μm thick slices.

| TTC staining
The cryostat was used to cut the frozen brain tissue into brain sections with a thickness of about 2 mm, which were placed in a tinfoil paper-coated culture dish for cassette operation.Subsequently, an appropriate amount of 2% 2,3,5-triphenyltetrazolium chlorides (Sigma Co.) was added to just immerse the sections, which was placed in a 37°C incubator for incubation for 30 min after covering the cassette lid.Then, the sections were washed with phosphate buffer saline (PBS) and fixed with 4% paraformaldehyde.The normal brain tissues were stained with bright red; if there is the infarction area, the area will not be stained and is pale due to decreased dehydrogenase activity.The images were imported into ImageJ software (version 1.52a, NIH) to quantify the cerebral infarction ratio and brain swelling.

| Immunofluorescence staining
Sections of brain tissue harvested in vivo were deparaffinized and tissue was encircled by a circle drawn with an immunohistochemical pen.Slices were rinsed three times with 0.01 M PBS (pH 7.4) for 5 min each at room temperature and subsequently preincubated with 0.3% Triton X-100 and 0.1% bovine serum albumin (BSA) for 1 h.After that, the prepared mixture of the TNF-α primary antibody (1:500, rabbit, Santacruz) was added to the sections and incubated overnight at 4°C.Tissue sections were rinsed three times for 5 min with 0.01 M PBS containing 0.1% Tween 20.Afterwards, sections were incubated with DayLight 594 antibody (1:200, anti-rabbit, Jackson) and counterstained for nuclei with DAPI.Finally, fluorescence microscope (DM4000B, Leica) at ×200 was used to acquire image of the hippocampus to observe the range of DAPI and TNF-α.

| Bioinformatics prediction
At the String website (https://cn.string-db.org/),TNF-α, NT-3, and TRKC were input into the multiple proteins mode, Rattus norvegicus was selected in organisms, and search was clicked to get the protein interaction network image which was exported to PNG format.

| Ventricular injection of effective TNF-α-shRNA lentivirus
To determine the effect of TNF-α on HIE rats, 3-day-old neonatal rats were anesthetized with 1.5%-2% isoflurane in oxygen-enriched air.Five microliters (2 × 10 8 /ml) of TNFα-shRNA (GCCCGTAGCCCACGTCGTCGTA) lentivirus (provided by RiboBio) was slowly injected into the right lateral ventricles at 1 μl/min under stereotactic guidance in the TNF-α-shRNA group (Digital stereotaxis Instrument with Fine Drive, My Neuro lab; coordinates: x = ± 0.5, y = +1.0,z = + 2.5 mm relative to bregma) and saline in the NC group. 21After 3 min, the tip was slowly pulled out and the skin was closed.To detect the inhibitory effect of the TNF-α-shRNA lentivirus injection, the expression of TNF-α was determined via RT-qPCR.

| RT-qPCR
The entire RNA of the fresh cortex, hippocampus, lung, and heart at 6, 12, and 24 h after HI modeling were isolated by RNAisoplus.Then, using the Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) and following the manufacturer's instructions, β-actin was amplified by reverse transcription of mRNA into complementary DNA (cDNA) using a RT-qPCR apparatus (CFX-96, Bio-rad).The primer sequences synthesized by Shuoqing Biotechnology Company were as follows: TNF-α: forward, GTTGGACC AATCATAGGCGC; reverse, CAATGTCG ATCACATGC ACCA; NT-3: forward, ACCGAACTCGAGTCCACCT; reverse, TGGAATTCTGACCTGGTGGC; TRKC: forward, GTCTGCAGCAAGACTGAGAT, reverse, CCAGTTCTCT ATGTGTCTGG; β-actin: forward, GAAGATCAAGATC ATTGCTCCT; and reverse, TACTCCTGCTTGCTGATCCA.The protocol of the variable-temperature heater was followed to carry out PCR: first, 95°C for 5 min, followed by 40 cycles of 95°C for 10 s, 51°C for 10 s, and 60°C for 20 s.The data were analyzed using the comparative critical threshold method with normalization of the β-actin value to 2 −ΔΔCt , which was used to determine the expression levels of TNF-α, NT-3, and TRKC.

| Statistical analysis
Mean ± standard deviation of the mean (SEM) and SPSS 25 (IBM Corp) were adopted to describe and analyze the experimental data, respectively.The normality test of numerical variables showed that they obeyed a normal distribution, so we used an independent-samples t-test for comparison between two groups and oneway analysis of variance (ANOVA) for comparison between multiple groups.The cerebral infarction ratio and brain swelling were analyzed using an independentsamples t-test and other data using one-way ANOVA methods according to variables.p < 0.05 was considered to indicate statistical significance.

| Successful establishment of an animal model of HIE
The Zea-Longa score in HIE injured rats increased compared to the sham group at 0, 4, 8, 12, and 24 h after surgery (Figure 1A, p < 0.05).The edema volume of the right brain of rats after the HI operation clearly increased compared with that of the sham group (Figure 1B,D, p < 0.05).The results of TTC staining of brain sections from SD rats showed that the volume of right cerebral infarction significantly increased after the HI operation (Figure 1C,E, p < 0.05).The above results showed that the neonatal rat HI model was successfully constructed.

| Expression of TNF-α upregulated in the lung, hippocampus, and cortex after the HI operation
We examined the expression of the inflammatory factor TNF-α following HIE.The results of RT-qPCR showed that the relative expression level of TNF-α showed clear upregulation after HI 6, 12, and 24 h in the hippocampus and the cortex, and also 24 h post HI in the lung when compared with the sham group (Figure 2A-C, p < 0.05), which indicated a severe inflammatory response after the HI operation.

| Localization of TNF-α in the hippocampus
We observed changes of TNF-α in the left and right hippocampus.The results of immunofluorescence staining indicated that TNF-α was expressed in the hippocampus, and its expression was higher in the right hippocampus (Figure 3).

| Correlation analysis between TNFα, NT-3, and TRKC
To further confirm the effect of TNF-α in brain injury following HIE, we selected NT-3 (encoded by the neurotrophin 3 [Ntf3]) and TRKC (encoded by the neurotrophin receptor tyrosine kinase [Ntrk3]), which are closely related to brain injury.Protein-protein interaction analysis indicated a direct or an indirect link among TNF-α, NT-3, and TRKC (Figure 4).Therefore, it is likely that TNFα plays a role in HIE through potential mechanisms that directly affect NT-3 and indirectly affect TRKC.

| Relative expression of NT-3, and TRKC upregulated after TNF-α interference
We verified whether TNF-α-shRNA lentivirus transfection was successful and detected expression of TNF-α, NT-3, and TRKC.TNF-α expression in the cortex and hippocampus of the TNF-α-shRNA group was significantly lower than that of the NC group after TNF-α interference, indicating successful ventricular injection of effective TNF-α-shRNA lentivirus (Figure 5A,B, p < 0.05).The expression levels of NT-3 in cortex and TRKC in cortex and heart were upregulated after TNF-α interference compared with the NC group in cortex and heart (Figure 5C,D,E, p < 0.05).

| DISCUSSION
In this study, we investigated the relationship between TNFα and NT-3, TRKC in neonatal HI rats, and the results showed that edema and increased infarct volume in the right brain occurred after HI modeling.Moreover, the relative expression level of TNF-α was increased in the neonatal rats after HI modeling.Furthermore, the expression of NT-3 and TRKC was found to be increased after TNF-α interference, which indicated that TNF-α interference ameliorates brain damage in neonatal hypoxic-ischemic encephalopathy rats via overexpression of NT-3 and TRKC.

| Increased cerebral infarction and edema after the HI operation
We found that the infarct volume and edema in the right brain after the HI operation were markedly increased compared to that in the sham group, which has been confirmed by previous studies. 22,23Breakdown of the blood-brain barrier leads to systemic leukocyte infiltration into the brain parenchyma and inflammation, further enhancing the formation of cerebral edema, which peaks at 24-48 h after the HI operation. 24ysregulation of the sodium and potassium pump causes additional calcium influx into cells, exacerbating negative effects such as ischemia and microvascular injury, | 385 leading to insufficient blood supply and an increase in cerebral infarction areas. 25Most brain tissue structures are paralyzed in functional areas due to brain edema and infarction injury caused by HI, which seriously endangers the life and health of newborns.

| Upregulation of NT-3 and TRKC after silencing TNF-α
In our study, it was discovered that the expression level of TNF-α was increased in the neonatal HI rats, which was consistent with previous studies. 26,27TNF-α is one of the key factors causing inflammation in a variety of diseases and can promote oxidative stress, necrosis, and apoptosis at the site of injury.Cerebral ischemia can induce an increase in both mRNA and protein expression levels of TNF-α in the lung tissues of rats with lung injury. 28It has also been reported that the expression of TNF-α in the hippocampus, cortex, and lung tissues gradually increased after brain damage. 29,30This indicates that TNF-α is one of the key factors in the pathomechanism of HIE.Some studies have found that the expression of proinflammatory factor TNF-α is upregulated in the hippocampus after nerve injury, while the expression of the brain-derived neurotrophic factor is downregulated, which shows a negative relationship. 31Moreover, TNF-α was significantly increased in ischemic retinal injury, while NT-3 expression was continuously and significantly downregulated, and there was a negative correlation between the two that is not reported in HIE. 32Then, after silencing TNF-α, the results showed that NT-3 and TRKC were upregulated in the HI condition.NT-3 is a protein encoded by the NTF3 gene in humans and synthesized in various tissues, and the protein encoded by the NT-3 gene is a neurotrophic factor in the family of neurotrophic protein NGF. 33,34ownregulation of cortical NT-3 expression has been reported in brain-related diseases. 35NT-3 could inhibit glial cell differentiation, improve the microenvironment of the lesion area, differentiate and survive existing neurons, promote endogenous nerve and brain tissue regeneration in previous study. 36RKC, a membrane-bound receptor for NT-3, is encoded by the Ntrk3 gene and mainly found in neurons, microglia, astrocytes, and endothelial cells in the central nervous system.Also, it can mediate the neuroprotective function of NT-3 and regulate neuronal survival, morphology, excitability, and neurite outgrowth by activating TRKC. 37,38Downregulation of cortical TRKC expression has been reported in brain-related diseases. 39Some studies have found that the inflammatory response produced by brain injury can affect cardiac function or even cause death, TRKC mRNA expression is significant in the heart, and NT-3 can reduce cardiomyocyte apoptosis to ameliorate the damage caused by myocardial ischemia/ reperfusion. 40,41It has been shown that interaction of NT-3 with TRKC can promote synaptogenesis of neural stem cell (NSC)-derived neurons in spinal cord injury. 42Similarly, it has been shown that the NT-3/ TRKC signaling pathway can ameliorate manganeseinduced neurotoxic effects and increase the survival of neurons. 43HIE is associated with heart rate disorders, and studies have found that the severity of neonatal hypoxic-ischemic encephalopathy can be reflected by heart rate variability (HRV). 44NT-3 and TRKC is associated with cardiac development and can maintain cardiovascular balance, which maybe improve the condition of HRV reduction caused by HIE. 45,46herefore, by silencing TNF-α, NT-3 and TRKC can be upregulated to promote the recovery of neurological function and other pathological lesions after HI injury, which maybe improve the mental retardation and movement disorders caused by brain injury.

| CONCLUSION
In summary, TNF-α interference may ameliorate brain dysfunction due to HIE by upregulating NT-3 and TRKC.At present, there are very few reports on the application of TRKC in HIE.Therefore, this provided further evidence to reveal the molecular mechanism of the action of TNF-α in neuronal injury after an HI operation.These findings maybe make TNF-α inhibitor a new way to ameliorate nerve damage caused by HIE.

AUTHOR CONTRIBUTIONS
Rong He contributed the central idea and conceived the entire experiment.Yong-Min Niu analyzed the data and wrote the first draft of the manuscript.Steven Z. Du helped to sort out ideas and provided guidance on writing the article.

F I G U R E 1
Changes in the Zea-Longa score, edema, and infarction in the right brain after the HI operation.(A) Changes in the Zea-Longa score after the HI operation compared to the sham group.(B, D) Changes in right brain edema after the HI operation compared to the sham group.(C, E) Changes in right cerebral infarction after the HI operation compared with the sham group.Data were described as mean ± SEM.HI, hypooxic-ischemic; h, hours.*p < 0.05.[Color figure can be viewed at wileyonlinelibrary.com]

F I G U R E 2
Relative expression of TNF-α after HI operation at 6 h, 12 h, and 24 h.(A-C) The relative expression of TNF-α was determined by RT-qPCR after the HI operation in the lung (A), hippocampus (B), and cortex (C).Data were described as mean ± SEM.HI, hypoxic-ischemic; h, hours.*p < 0.05.F I G U R E 3 Immunofluorescent of TNF-α in the hippocampus.(A, B) The localization of TNF-α was detected by immunofluorescence in the hippocampus.Sections were stained with DAPI (blue, the first panel) to show the nucleus of all cells, TNF-α (green, the second panel), and the merged images (the last panel).Scale bars, 100 μm, 50 μm for the enlarged area.Hippo-right, hippocampus right; Hippo-left, hippocampus left.[Color figure can be viewed at wileyonlinelibrary.com]NIU ET AL.

F I G U R E 4
Protein interaction analysis of TNF-α, NT-3 and TRKC.The red lines and the blue line represent known interactions between proteins that have been experimentally determined and from curated databases, respectively; the green lines represent text mining.[Color figure can be viewed at wileyonlinelibrary.com]F I G U R E 5 Effect on the expression of TNF-α, NT-3 and TRKC after interfering TNF-α in vivo.(A) Compared with the NC group, the relative expression changes of TNF-α in the right hippocampus in the TNF-α-shRNA group.(B, C, D) Compared with the NC group, the relative expression changes of TNF-α, NT-3, and TRKC in the right cortex in the TNF-α-shRNA group.(E) Relative expression changes of TRKC in the heart compared with the NC group.Data were described as mean ± SEM. *p < 0.05.