A Novel Netrin-1-Derived Peptide Enhances Protection Against Neuronal Death And Mitigates Of Intracerebral Hemorrhage in Mice Part 3

Treatment groups of saline, TAT, or TE1 (dissolved in saline) were given to mice 2 h after ICH and every day for 5 days by intraperitoneal injection at a total volume of 0.1 mL per animal. Peptides were given in a blinded manner at doses of 12 mg/kg.

Physiological saline is a commonly used physiological buffer, widely used in medical and biological experiments. But did you know? Physiological saline can also improve our memory!

First of all, physiological saline plays an important role in the normal function of cells. It can maintain normal cell osmotic pressure and maintain the normal morphology and function of cells. In the brain, a large number of nerve cells need physiological saline as a basic substance, and the supplement of physiological saline can promote the normal operation of brain cells, provide sufficient oxygen and nutrients for the brain, and make the brain healthier.

Secondly, physiological saline can promote signal transmission between brain neurons and enhance the learning ability of the brain. Studies have shown that in the process of task learning, the injection of physiological saline can significantly improve the learning effect of learners, and improve the learners' emotional state, reduce learning anxiety and stress. At the same time, physiological saline can also increase the number of neurons and the complexity of neurons in the hippocampus, which is conducive to the formation and maintenance of memory.

In addition, physiological saline can improve the metabolic environment of the brain and reduce various harmful effects on the brain, such as damage, lesions, ischemia, and hypoxia. These are factors that can hurt the brain, and the injection of physiological saline can enhance the brain's coping ability and reduce its damage, thereby improving the brain's memory and thinking ability.

In general, saline is an ideal substance for improving memory. It can have multiple positive effects on the brain, including increasing the number and complexity of neurons, enhancing the metabolic environment, and promoting signal transmission between neurons. Therefore, when we need to improve our memory, we might as well consider injecting saline to provide better protection and support for our brain. It can be seen that we need to improve our memory, and Cistanche can significantly improve our memory because it can also regulate the balance of neurotransmitters, such as increasing the levels of acetylcholine and growth factors, which are very important for memory and learning. In addition, Cistanche can also improve blood flow and promote oxygen delivery, which can ensure that the brain obtains sufficient nutrition and energy, thereby improving brain vitality and endurance.

Click know ways to improve your memory

4.13. Collagenase-Induced Mouse Model of ICH

Collagenase injection is commonly used to induce ICH [60]. Male C57BL/6 mice (8 to 12 weeks of age) were anesthetized with isoflurane (2 to 5%) and placed on a stereotaxic frame. 

During the procedure, the body temperature of each animal was maintained at 37 ◦C with a homeothermic blanket. Using a stereotaxic instrument (Ruiwode Life Science Co., Ltd., Shenzhen, China) and a syringe (Hamilton Company, Reno, NV, USA), 0.3 µL of collagenase (0.045 IU; Sigma-Aldrich, St Louis, MO, USA) was infused into the right striatum (AP: +0.2 mm; ML: −2.3 mm; DV: −3.5 mm) at a flow rate of 0.1 µL/min. 

A total of 0.3 µL of saline was infused into control animals. All surgical procedures were conducted under aseptic conditions. In this study, over 66 male mice were used, and the animals were randomly assigned to groups before each experiment [57].

4.14. Behavioral Analysis

The neurological behavioral analysis of mice after brain hematoma formation was detected by the adhesive tape removal test [61] and cylinder test [60]. The adhesive tape removal test was performed by placing the adhesive tape on the planter region of each forepaw (right and left) of the mice. 

Each mouse was placed in a novel cage. 

The time from when the tape was applied to when the mouse successfully removed it was recorded for each paw. A maximum of 300 s was allowed for each paw. Between experiments, the animals were housed in their home cages in a pathogen-free facility. 

The cylinder test was performed by placing a mouse in a transparent acrylic glass cylinder (diameter: 8 cm; height: 25 cm) in front of 2 mirrors and a camera for 5 min. The camera was placed centrally in front of the 2 mirrors and the cylinder to obtain an optimal video. 

For assessment of independent forelimb use, (1) the ability of the mice to contact the cylinder wall with one forelimb during full rearing and (2) the ability of the mice to land with only one forelimb on the floor after full rearing were evaluated. At least 20 contact points for each forelimb were counted using slow motion. 

For baseline analysis before surgery, the test was performed twice per mouse with a 1 h break between trials. Forelimb use is expressed as the ratio of right- to left-sided independent forelimb use.

4.15. Haematoma Volume Analysis

To determine if the treatments reduced the hematoma volume, mice were sacrificed 5 days after ICH; the brains were removed and flash frozen in OCT. Coronal sections were sliced at a thickness of 30 µm and placed directly on glass slides. To quantify the hematoma volume, the sections were digitized at standardized coronal levels. 

A blinded user measured the contours and hematoma in the left and right hemispheres in each section. The hematoma volume and swelling were measured and calculated using ImageJ (NIH). 

At least 5 brains per group were subjected to hematoma measurements, and the hematoma volume was measured throughout the brain.

4.16. Fluoro-Jade C Staining

Neurodegeneration in mice following collagenase-induced ICH was assessed by staining with a Fluoro-jade C Ready-to-Dilute Staining Kit (TR-100-FJT, Biosensis, Thebarton, Australia) according to the manufacturer's protocol [62]. 

Briefly, control mice and ICH mice (±i.p. peptide) were sacrificed 5 days after modeling, and the brains were processed according to the protocol used for the immunocytochemistry experiment. 

Floating brain sections were immersed in 1% sodium hydroxide solution for 5 min, rinsed for 2 min in 70% ethanol, and then rinsed for 2 min in distilled water. Next, the floating sections were immersed in a 0.06% potassium permanganate solution (KMnO4 in distilled water) for 10 min. 

The sections were washed with ddH2O before being immersed in 0.0001% Fluoro-jade staining solution (Fluoro-jade C in distilled water) for 20 min with gentle shaking in the dark. 

The sections were washed with ddH2O, mounted on coated slides, and dried at room temperature overnight in the dark. Fluoro-Jade C staining within the perihematomal region was examined using a Zeiss LSM 880 confocal microscope. Quantitation of Fluoro-jade C staining was performed using ImageJ software.

4.17. Tissue Immunofluorescence

Tissue immunofluorescence staining was performed using a series of 30-µm-thick sections. Sections were incubated with the following primary antibodies: mouse antiNeuN (1:500, Millipore, MAB377, Billerica, MA, USA) or mouse anti-GFAP (1:200, Sigma Aldrich, G3893, St Louis, MO, USA) antibodies at 4 ◦C overnight. After washing with PBS, sections were then incubated with Alexa Fluor 488-conjugated donkey anti-mouse (1:1000; Invitrogen, A21202, Carlsbad, CA, USA) antibody.

4.18. TUNEL Assay

A terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL) assay was used to identify apoptotic cells with nuclear DNA fragmentation in the brain perihematomal region. 

Staining was done using a commercial one-step TUNEL apoptosis assay kit (Beyotime, C1090, Nanjing, China) and performed according to the manufacturer's instructions. Following tissue immunofluorescence, the TUNEL reaction was performed. 

Brain sections were incubated with a TUNEL reaction mixture (1 h at 37 ◦C), and then sections were washed with PBS and counterstained with DAPI. The co-localization of NeuN-TUNEL staining was examined using a Zeiss LSM 880 confocal microscope. 

TUNEL-positive neurons (NeuN positive) in the perihematomal region inside the striatum were quantified using ImageJ software on three different pictures per mouse taken under a 20X objective. The colocalization of GFAP-TUNEL staining was similar to that of NeuN-TUNEL staining.

4.19. In Vivo Blood-Brain Barrier Permeability Assay

In Vivo, Blood-brain Barrier Permeability Assays were performed as described previously [63–65]. Briefly, control mice and ICH mice (±i.p. peptide) were intraperitoneally injected with FITC-labelled 4 kDa dextran (100 µL of 2 mM stock in PBS, Sigma Aldrich, 46944-100 MG-F, St Louis, MO, USA) after modeling 5 days, followed by anesthesia 5 min later. 

After a circulation time of 10 min for the tracer, the animals were prepared for transcardial perfusion, and ~ 300 µl blood was collected from the chest cavity just after atrial puncture, followed by perfusion for 3 min with PBS. Hemibrain (free of the olfactory lobes, cerebellum, and hindbrain) and a single kidney were collected and immediately frozen in liquid nitrogen and stored at −80 ◦C. 

For fluorescence measurement, samples were thawed on ice, weighed, and homogenized in PBS (400 µL for hemicerebrum and 600 µL for kidney), followed by centrifugation for 20 min at 4 ◦C and 15,000× g. 

Supernatants (100 µL), as well as equal volumes of serum (1/5 dilution in PBS), were loaded into a 96-well black plate, and fluorescence was measured at the corresponding excitation (490 nm)/emission (520 nm) in a plate reader (Tecan, Männedorf, Switzerland). Sham animals (without tracer injection) were used to subtract autofluorescence values. The permeability index (mL/g) was calculated as the ratio of tissue RFUs/g tissue weight to serum RFUs/mL serum.

4.20. Fluorescent Lectin Staining

To visualize brain microvessels, we used fluorescein-conjugated Lycopersicon esculentum lectin (1:200, Vector Laboratories, FL-1171, Burlingame, CA, USA) to immerse floating sections for 1 h. 

Sections were imaged on a Zeiss LSM 880 confocal microscope under a 20× objective, and images were processed with a 1.3 gamma setting. Three images were taken from 3 prespecified areas in the perihematomal region of the coronal brain per animal. Vessel branch points were quantified in each brain by a blinded investigator. A branch point was defined as a single fluorescein-labeled vessel noticeably separating into 2 distinct and separate vessel tracks [66].

4.21. Statistical Analysis

All experimental procedures were conducted blinded and performed 3 times independently as previously described [67]. Data are presented as the mean ± SEM. Statistical analysis was performed by One-way ANOVA, Two-way ANOVA, and Student's t-test, as appropriate, using GraphPad Prism v7 (GraphPad Software Inc., San Diego, CA, USA) and SPSS software version 20.0 (SPSS, Inc., Chicago, IL, USA). 

The statistical parameters can be found in the relevant figure legends. Statistical significance was set at * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.]

5. Conclusions

The EGF3 domain of Netrin-1 is crucial to the interaction of Netrin-1 with DCC and can activate different signaling pathways. Truncated peptide E1 of Netrin-1 (residues 407–422) interacts with DCC to protect neurons from hemin-induced toxicity. 

The application of peptide E1 improves functional recovery in an experimental model of ICH. Furthermore, peptide E1 can protect neurons from degeneration and apoptosis after ICH.

Author Contributions: Conceptualization, L.L., Z.-X.H. and X.-J.Z.; methodology, L.L., K.-J.L. and J.-B.C.; validation H.-L.Y. and X.-X.H.; investigation, X.-J.Z.; data curation, J.Y.; resources L.L. and Z.-X.H.; writing L.L., Z.-X.H. and X.-J.Z.; supervision, Z.-X.H., X.-J.Z.; funding acquisition, Z.-X.H. and X.-J.Z. All authors have read and agreed to the published version of the manuscript.

Funding: This research was funded by the National Natural Science Foundation of China (31671065 and 32071018), Science and Technology Development Plan of Jilin Province (202520YY01017404).

Institutional Review Board Statement: All procedures were approved by the Institutional Animal Care and Use Committee of Northeast Normal University (NENU/IACUC, AP2016200299). National Standards of the People's Republic of China (GB/T 35892–2018), Laboratory Animal-Guideline for Ethical Review of Animal Welfare, was the guidance for our animal care and protocols.

Informed Consent Statement: Not applicable.

Acknowledgments: This work was supported by the National Natural Science Foundation of China (31671065 and 32071018) and, the Science and Technology Development Plan of Jilin Province (202520YY01017404).

Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References

1. Kirshner, H.S. Medical management of intracerebral hemorrhage: Back to the basics. Int. J. Clin. Pract. 2008, 62, 521–522. [CrossRef] [PubMed] 

2. Anderson, C.S.; Heeley, E.; Huang, Y.; Wang, J.; Stapf, C.; Delcourt, C.; Lindley, R.; Robinson, T.; Lavados, P.; Neal, B.; et al. Rapid blood pressure lowering in patients with acute intracerebral hemorrhage. N. Engl. J. Med. 2013, 368, 2355–2365. [CrossRef] [PubMed] 

3. Qureshi, A.I.; Mendelow, A.D.; Hanley, D.F. Intracerebral hemorrhage. Lancet (Lond. Engl.) 2009, 373, 1632–1644. [CrossRef] 

4. Keep, R.F.; Hua, Y.; Xi, G. Intracerebral hemorrhage: Mechanisms of injury and therapeutic targets. Lancet Neurol. 2012, 11, 720–731. [CrossRef] 

5. Schrag, M.; Kirshner, H. Management of Intracerebral Hemorrhage: JACC Focus Seminar. J. Am. Coll. Cardiol. 2020, 75, 1819–1831. [CrossRef] [PubMed] 

6. Kandasamy, R.; Idris, Z.; Abdullah, J.M. Surgery of Intracerebral Hemorrhage. In Neurovascular Surgery Surgical Approaches for Neurovascular Diseases; July, J., Wahjoepramono, E.J., Eds.; Springer: Singapore, 2019; pp. 201–210. 7. Broughton,

7. Broughton, B.R.; Reutens, D.C.; Sobey, C.G. Apoptotic mechanisms after cerebral ischemia. Stroke 2009, 40, e331–e339. [CrossRef] [PubMed]

8. Kristiansen, M.; Graversen, J.H.; Jacobsen, C.; Sonne, O.; Hoffman, H.J.; Law, S.K.; Moestrup, S.K. Identification of the haemoglobin scavenger receptor. Nature 2001, 409, 198–201. [CrossRef] 

9. Madangarli, N.; Bonsack, F.; Dasari, R.; Sukumari-Ramesh, S. Intracerebral Hemorrhage: Blood Components and Neurotoxicity. Brain Sci. 2019, 9, 316. [CrossRef] 

10. Deiner, M.S.; Kennedy, T.E.; Fazeli, A.; Serafini, T.; Tessier-Lavigne, M.; Sretavan, D.W. Netrin-1 and DCC mediate axon guidance locally at the optic disc: Loss of function leads to optic nerve hypoplasia. Neuron 1997, 19, 575–589. [CrossRef] 

11. Kennedy, T.E.; Serafini, T.; de la Torre, J.R.; Tessier-Lavigne, M. Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord. Cell 1994, 78, 425–435. [CrossRef] 

12. Flores, C. Role of netrin-1 in the organization and function of the mesocorticolimbic dopamine system. J. Psychiatry Neurosci. Jpn. 2011, 36, 296–310. [CrossRef]

For more information:1950477648nn@gmail.com