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<front>
<journal-meta>
<journal-id journal-id-type="publisher-id">Front. Cell. Neurosci.</journal-id>
<journal-title>Frontiers in Cellular Neuroscience</journal-title>
<abbrev-journal-title abbrev-type="pubmed">Front. Cell. Neurosci.</abbrev-journal-title>
<issn pub-type="epub">1662-5102</issn>
<publisher>
<publisher-name>Frontiers Media S.A.</publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id pub-id-type="doi">10.3389/fncel.2024.1344853</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Cellular Neuroscience</subject>
<subj-group>
<subject>Brief Research Report</subject>
</subj-group>
</subj-group>
</article-categories>
<title-group>
<article-title>Cell-free fat extract promotes axon regeneration and retinal ganglion cells survival in traumatic optic neuropathy</article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author" equal-contrib="yes">
<name>
<surname>Sun</surname>
<given-names>Yiyu</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>1</sup></xref>
<xref ref-type="author-notes" rid="fn0004"><sup>&#x2020;</sup></xref>
<uri xlink:href="https://loop.frontiersin.org/people/1516280/overview"/>
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<role content-type="https://credit.niso.org/contributor-roles/writing-original-draft/"/>
</contrib>
<contrib contrib-type="author" equal-contrib="yes">
<name>
<surname>Chen</surname>
<given-names>Di</given-names>
</name>
<xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
<xref ref-type="author-notes" rid="fn0004"><sup>&#x2020;</sup></xref>
<uri xlink:href="https://loop.frontiersin.org/people/1494840/overview"/>
<role content-type="https://credit.niso.org/contributor-roles/data-curation/"/>
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</contrib>
<contrib contrib-type="author">
<name>
<surname>Dai</surname>
<given-names>Tao</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>1</sup></xref>
<role content-type="https://credit.niso.org/contributor-roles/software/"/>
<role content-type="https://credit.niso.org/contributor-roles/writing-review-editing/"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Yu</surname>
<given-names>Ziyou</given-names>
</name>
<xref ref-type="aff" rid="aff3"><sup>3</sup></xref>
<role content-type="https://credit.niso.org/contributor-roles/software/"/>
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</contrib>
<contrib contrib-type="author">
<name>
<surname>Xie</surname>
<given-names>Hui</given-names>
</name>
<xref ref-type="aff" rid="aff4"><sup>4</sup></xref>
</contrib>
<contrib contrib-type="author" corresp="yes">
<name>
<surname>Wang</surname>
<given-names>Xiangsheng</given-names>
</name>
<xref ref-type="aff" rid="aff5"><sup>5</sup></xref>
<xref ref-type="corresp" rid="c001"><sup>&#x002A;</sup></xref>
<role content-type="https://credit.niso.org/contributor-roles/conceptualization/"/>
<role content-type="https://credit.niso.org/contributor-roles/writing-original-draft/"/>
</contrib>
<contrib contrib-type="author" corresp="yes">
<name>
<surname>Zhang</surname>
<given-names>Wenjie</given-names>
</name>
<xref ref-type="aff" rid="aff3"><sup>3</sup></xref>
<xref ref-type="corresp" rid="c002"><sup>&#x002A;</sup></xref>
<uri xlink:href="https://loop.frontiersin.org/people/1836944/overview"/>
<role content-type="https://credit.niso.org/contributor-roles/conceptualization/"/>
<role content-type="https://credit.niso.org/contributor-roles/funding-acquisition/"/>
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</contrib-group>
<aff id="aff1"><sup>1</sup><institution>Department of Wound Reconstructive Surgery, Tongji Hospital Affiliated to Tongji University, School of Medicine, Tongji University</institution>, <addr-line>Shanghai</addr-line>, <country>China</country></aff>
<aff id="aff2"><sup>2</sup><institution>Department of Neurosurgery, The First Affiliated Hospital of Zhengzhou University</institution>, <addr-line>Zhengzhou, Henan</addr-line>, <country>China</country></aff>
<aff id="aff3"><sup>3</sup><institution>Shanghai Key Laboratory of Tissue Engineering, Department of Plastic and Reconstructive Surgery, Shanghai 9th People&#x2019;s Hospital, Shanghai Jiao Tong University</institution>, <addr-line>Shanghai</addr-line>, <country>China</country></aff>
<aff id="aff4"><sup>4</sup><institution>Department of Geriatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology</institution>, <addr-line>Wuhan, Hubei</addr-line>, <country>China</country></aff>
<aff id="aff5"><sup>5</sup><institution>Department of Plastic and Reconstructive Surgery, Affiliated Hangzhou First People&#x2019;s Hospital, School of Medicine, Westlake University</institution>, <addr-line>Hangzhou, Zhejiang</addr-line>, <country>China</country></aff>
<author-notes>
<fn fn-type="edited-by" id="fn0005">
<p>Edited by: Clara Penas, Autonomous University of Barcelona, Spain</p>
</fn>
<fn fn-type="edited-by" id="fn0006">
<p>Reviewed by: Yingjie Wang, Nantong University, China</p>
<p>Simone Di Giovanni, Imperial College London, United Kingdom</p>
</fn>
<corresp id="c001">&#x002A;Correspondence: Xiangsheng Wang, <email>doctorwxs@yeah.net</email></corresp>
<corresp id="c002">Wenjie Zhang, <email>wenjieboshi@aliyun.com</email></corresp>
<fn fn-type="equal" id="fn0004">
<p><sup>&#x2020;</sup>These authors have contributed equally to this work and share first authorship</p>
</fn>
</author-notes>
<pub-date pub-type="epub">
<day>07</day>
<month>03</month>
<year>2024</year>
</pub-date>
<pub-date pub-type="collection">
<year>2024</year>
</pub-date>
<volume>18</volume>
<elocation-id>1344853</elocation-id>
<history>
<date date-type="received">
<day>26</day>
<month>11</month>
<year>2023</year>
</date>
<date date-type="accepted">
<day>27</day>
<month>02</month>
<year>2024</year>
</date>
</history>
<permissions>
<copyright-statement>Copyright &#x00A9; 2024 Sun, Chen, Dai, Yu, Xie, Wang and Zhang.</copyright-statement>
<copyright-year>2024</copyright-year>
<copyright-holder>Sun, Chen, Dai, Yu, Xie, Wang and Zhang</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/">
<p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</p>
</license>
</permissions>
<abstract>
<p>Injuries to axons within the central nervous system (CNS) pose a substantial clinical challenge due to their limited regenerative capacity. This study investigates the therapeutic potential of Cell-free fat extract (CEFFE) in CNS injury. CEFFE was injected intravitreally after the optic nerve was crushed. Two weeks post-injury, quantification of regenerated axons and survival rates of retinal ganglion cells (RGCs) were performed. Subsequently, comprehensive gene ontology (GO) an-notation elucidated the cellular origins and functional attributes of CEFFE components. Molecular mechanisms underlying CEFFE&#x2019;s therapeutic effects were explored through Western blotting (WB). Additionally, levels of inflammatory factors within CEFFE were determined using enzyme-linked immunosorbent assay (ELISA), and histological staining of microglia was conducted to assess its impact on neuroinflammation. CEFFE demonstrated a significant capacity to promote axon re-generation and enhance RGCs survival. GO annotation revealed the involvement of 146 proteins within CEFFE in axonogenesis and neurogenesis. WB analysis unveiled the multifaceted pathways through which CEFFE exerts its therapeutic effects. Elevated levels of inflammatory factors were detected through ELISA, and CEFFE exhibited a modulatory effect on microglial activation in the retinal tissue following optic nerve crush (ONC). The present study highlights the therapeutic promise of CEFFE in the management of CNS injuries, exemplified by its ability to foster axon regeneration and improve RGCs survival.</p>
</abstract>
<kwd-group>
<kwd>cell-free fat extract</kwd>
<kwd>axon regeneration</kwd>
<kwd>RGCs survival</kwd>
<kwd>inflammation factors</kwd>
<kwd>microglia</kwd>
</kwd-group>
<counts>
<fig-count count="5"/>
<table-count count="1"/>
<equation-count count="0"/>
<ref-count count="45"/>
<page-count count="10"/>
<word-count count="6372"/>
</counts>
<custom-meta-wrap>
<custom-meta>
<meta-name>section-at-acceptance</meta-name>
<meta-value>Cellular Neuropathology</meta-value>
</custom-meta>
</custom-meta-wrap>
</article-meta>
</front>
<body>
<sec sec-type="intro" id="sec1">
<label>1</label>
<title>Introduction</title>
<p>The axon serves as a foundational structural and functional unit of a neuron, pivotal in the transmission of signals and substances throughout the body. After CNS injury, lesioned axons fail to regenerate, which leads to compromised functional recovery (<xref ref-type="bibr" rid="ref9">Fischer and Leibinger, 2012</xref>; <xref ref-type="bibr" rid="ref4">Blennow et al., 2016</xref>). These challenges can manifest as conditions such as blindness or paralysis. The optic nerve crush (ONC) model is one of most predominant method to simulate axon injury in CNS&#x2014;given that the retina is categorically a part of the CNS (<xref ref-type="bibr" rid="ref11">Gennarelli et al., 1989</xref>; <xref ref-type="bibr" rid="ref43">Williams et al., 2020</xref>). More importantly, the mechanism of injury has been verified by the optic nerve crush model and translated to other axon injury models, including those of spinal cord injury.</p>
<p>In recent years stem cells have been shown to promote axon regeneration (<xref ref-type="bibr" rid="ref32">Mesentier-Louro et al., 2014</xref>; <xref ref-type="bibr" rid="ref36">Park et al., 2021</xref>). Among various stem cell classifications, adipose-derived stem cells (ADSCs) are favored because of their accessibility, reduced immunogenicity, and multiple differentiation potential. Stem cells may facilitate therapeutic applications by both supplanting native cells and discharging trophic factors. Yet, existing literature lacks robust evidence to suggest that ADSCs can differentiate directly into RGCs. ADSCs contain many trophic factors, such as the vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF) and hepatocyte growth factor (HGF). Exogenously applied trophic factors, such as Ciliary neurotrophic factor (CNTF) or HGF, have been observed to stimulate regeneration in injured axons. Concurrently, certain studies suggest that the synergistic application of multiple trophic factors yields superior outcomes compared to monotherapeutic approaches (<xref ref-type="bibr" rid="ref25">Logan et al., 2006</xref>).</p>
<p>In prior investigations, we pioneered a novel methodology to generate cell-free fat extract (CEFFE). CEFFE is an acellular fluid extracted from human subcutaneous adipose tissue without cellular components and lipid residues and has shown no immunogenicity, good biosafety. Our previous research has demonstrated the robust potential of CEFFE in tissue repair and regeneration, such as skeletal, skin and reproductive systems (<xref ref-type="bibr" rid="ref8">Deng et al., 2020</xref>; <xref ref-type="bibr" rid="ref15">Jia et al., 2022</xref>; <xref ref-type="bibr" rid="ref24">Liu et al., 2022</xref>). However, the effect of CEFFE on CNS injury remains unknown. Since the composition of CEFFE is similar to stem cell paracrine, we hypothesized that CEFFE might possess the potential to promote axon regeneration akin to stem cells. To test this hypothesis, this study evaluated the effects of CEFFE on axon regeneration in ONC model and investigated the underlying mechanisms.</p>
</sec>
<sec sec-type="materials|methods" id="sec2">
<label>2</label>
<title>Materials and methods</title>
<sec id="sec3">
<label>2.1</label>
<title>CEFFE preparation</title>
<p>The preparation of the CEFFE adhered to established protocols (<xref ref-type="bibr" rid="ref45">Yu et al., 2018</xref>; <xref ref-type="bibr" rid="ref8">Deng et al., 2020</xref>; <xref ref-type="bibr" rid="ref44">Xu et al., 2020</xref>). Briefly, human adipose tissue was acquired from healthy liposuction donors. The tissue was rinsed with saline to remove red blood cells and centrifuged at 1200&#x2009;&#x00D7;&#x2009;<italic>g</italic> for 3&#x2009;min. The upper oily and lower fluid layers were then discarded, and the middle fat layer was harvested and mechanically emulsified. The emulsified fat was then frozen at &#x2212;80&#x00B0;C for 24&#x2009;h and thawed at 37&#x00B0;C for 30&#x2009;min to further disrupt the cells in the fat tissue. After one cycle of the freeze/thaw process, the fat was centrifuged again at 2000&#x2009;&#x00D7;&#x2009;<italic>g</italic> for 5&#x2009;min and separated into four layers, and the third aqueous layer containing the CEFFE was collected and frozen at &#x2212;80&#x00B0;C for experimental purposes. The final extract was passed through a 0.22-&#x03BC;m filter (Corning Glass Works, Corning, NY, USA) for sterilization and removal of cell debris. The CEFFE protein concentrations were measured, using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). The study was approved by the Ethics Committee of the Shanghai Jiaotong University School of Medicine, Shanghai, China (SH9H-2018-T22-1).</p>
</sec>
<sec id="sec4">
<label>2.2</label>
<title>Surgical procedures</title>
<p>C57 BL/6 mice aged 5&#x2009;weeks were used in this experiment. All surgical procedures were conducted under anesthesia, with 1% pentobarbital sodium (10&#x2009;&#x03BC;l/g) administered intraperitoneally. After surgery, ointment containing ophthalmic antibiotic was applied to protect the cornea. Mice were kept on a 12-h light/dark cycle with free access to food and water. Mice with opaque lenses were excluded from the experiment. The study was approved by the Ethics Committee of the Shanghai Jiaotong University School of Medicine, Shanghai, China.</p>
<p>Subsequent to anesthesia administration, the left optic nerve was exposed intraorbitally. The optic nerve was crushed with forceps (Fine Science Tools, 11480-11) for 5&#x2009;s, 1&#x2009;mm distal to the eyeball. Care was taken to avoid damaging the underlying ophthalmic arteries. Immediately after crushing, a fine glass micropipette was inserted into the posterior chamber of the left eye and avoid damaging the lens. Before injection, approximately 3&#x2009;&#x03BC;l vitreous humor was pumped out. Subsequently, using a Nanoliter 2010 injector (WPI, NL2010MC2T), 3&#x2009;&#x03BC;l of the CEFFE (5&#x2009;&#x03BC;g/&#x03BC;l), Phosphate buffered saline (PBS) or CNTF (5&#x2009;&#x03BC;g/&#x03BC;l, R&#x0026;D system, 257-NT-050) were slowly injected into the vitreous chamber at the appropriate time points, in accordance with the needs of the experiments. The needle was removed after 5&#x2009;min until the solution completely diffused.</p>
<p>To label regenerating axons, 2&#x2009;&#x03BC;l of Alexa-555 conjugated cholera toxin &#x03B2; subunit (CTB-555) (2&#x2009;&#x03BC;g/&#x03BC;l, C22843, Thermo Fisher) was injected into the vitreous with a Hamilton syringe (80,030, Hamilton) 2&#x2009;weeks later after the initial injury.</p>
</sec>
<sec id="sec5">
<label>2.3</label>
<title>Immunohistochemistry</title>
<p>Mice were humanely sacrificed and intracardially perfused with a cold saline solution, followed by a PBS solution containing 4% Paraformaldehyde (PFA). The eyes and optic nerves were carefully removed from the connective tissue, postfixed for 2&#x2009;h in 4% PFA solution at 4&#x00B0;C, and subsequently transferred to 30% sucrose for a 48-h incubation at 4&#x00B0;C. For section staining, the retinal sections were incubated with primary antibodies CD11b (1:200, Abcam ab226482) overnight at 4&#x00B0;C and washed three times for 15&#x2009;min each with PBS. Secondary antibodies were then applied and incubated for 1&#x2009;h at room temperature. For whole-mount retina staining, the retina was cut into petals and blocked in PBS containing 5% normal goat serum and 0.4% Triton X-100 for 2&#x2009;h at room temperature. Thereafter, the tissues were incubated with primary antibody Neuronal Class III &#x03B2;-Tubulin (Tuj1) (1:500, Abcam ab18207) in a blocking buffer overnight at 4&#x00B0;C. After three rinses with PBST (1x Phosphate-Buffered Saline, 0.1% Tween 20), retinas were incubated with the secondary antibody goat anti-rabbit Alex-488 (1:1000 dilution, Thermo Fisher R37116). Following three washes with PBST, the tissues were sealed for observation. The optic nerve was embedded in O.C.T. Compound (25608&#x2013;930, SAKURA) and longitudinal sections (14&#x2009;&#x03BC;m) were prepared and stored at &#x2212;20&#x00B0;C until further use.</p>
</sec>
<sec id="sec6">
<label>2.4</label>
<title>Quantification of axons, RGCs and microglia</title>
<p>The number of CTB-labeled axons was counted at different distances from the crush site (250, 500, 750, 1,000, and 1,500&#x2009;&#x03BC;m) in four sections per nerve. Each group included six mice. The cross-sectional width of the nerve was measured at the point at which the counts were taken and used to calculate the number of axons per millimeter of nerve width. The number of axons per millimeter was then averaged over all sections. In addition, &#x03A3;a<sub>d</sub>, the total number of axons extending distance d in a nerve with radius r, was estimated by summing over all sections with thickness t (14&#x2009;&#x03BC;m), as follows (<xref ref-type="bibr" rid="ref37">Park et al., 2008</xref>): &#x03A3;a<sub>d</sub>&#x2009;=&#x2009;&#x03C0;r<sup>2</sup>&#x2009;x [average axons/mm]/t.</p>
<p>For RGCs counting, whole mount retinas were immunostained with the Tuj1 antibody and six to nine fields were randomly sampled from each retina. The percentage of RGCs survival was calculated as the ratio of the number of surviving RGCs in the crushed eyes to that in the contralateral uninjured eyes. Counts of surviving cells and regenerated axons were performed by a single observer who was blinded to the surgical manipulations and treatments.</p>
<p>For microglia quantification, the magnitude of CD11b&#x2009;+&#x2009;microglia activation was defined as the number of CD11b&#x2009;+&#x2009;cells present in the total area. Four to five slices of retina per mouse were randomly selected to count the total number of CD11b&#x2009;+&#x2009;cells and the number of CD11b&#x2009;+&#x2009;cells present in the GCL (ganglion cell layer) layer.</p>
</sec>
<sec id="sec7">
<label>2.5</label>
<title>Proteomics analysis</title>
<p>The CEFFE samples were processed for quantitative proteomic analysis as previously described (<xref ref-type="bibr" rid="ref45">Yu et al., 2018</xref>). Briefly, protein concentration of CEFFE was measured, using a BCA protein assay kit. The samples were then digested with trypsin for subsequent Liquid chromatography&#x2013;tandem mass spectrometry analysis (LC-MC). Gene ontology (GO) analysis was performed to classify all identified protein types into three categories (cell component, molecular function, and biological process), using the UniProt-GOA database,<xref ref-type="fn" rid="fn0001"><sup>1</sup></xref> InterProScan,<xref ref-type="fn" rid="fn0002"><sup>2</sup></xref> and GO annotation.<xref ref-type="fn" rid="fn0003"><sup>3</sup></xref> Proteins related to axon genesis and neurogenesis were also identified.</p>
</sec>
<sec id="sec8">
<label>2.6</label>
<title>Western blot assays</title>
<p>Three mice per condition were sacrificed by cervical dislocation and retinas were dissected, homogenized, and solubilized at 4&#x00B0;C in a cell lysis buffer (P0013, Beyotime) for 30&#x2009;min, supplemented with 1&#x2009;mM phenylmethylsulfonyl fluoride, 50&#x2009;mM sodium fluoride, 1&#x2009;mM Na3VO4, and a protease inhibitor. Total protein lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and analyzed by western blotting with anti-mTOR (Merck SAB2702297, dilution 1:1000), anti-ROCK2 (Santa Cruz sc-398519, dilution 1:1000), anti-cleaved calpain (Abcam ab92333, 1:1000) and anti-&#x03B2;-actin (Beyotime AF5001, 1:1000). Data analysis was performed using the NIH ImageJ software. The mean density of each band was normalized to that of the &#x03B2;-actin signal in the same sample.</p>
</sec>
<sec id="sec9">
<label>2.7</label>
<title>Enzyme-linked immunosorbent assay</title>
<p>Enzyme-linked immunosorbent assays (R&#x0026;D Systems company, USA) were performed, according to standard protocols, to quantify the levels of transforming growth factor beta 1 (TGF&#x03B2;-1), tumor necrosis factor alpha (TNF&#x03B1;), interleukin 4 (IL-4), and interleukin 1 beta (IL-1&#x03B2;) in CEFFE. The antibodies used were as followed: Human/Mouse/Rat/Porcine/Canine TGF-beta 1 Quantikine ELISA, Catalog #: DB100C; Human IL-4 Quantikine HS ELISA Kit, Catalog #: HS400; Human IL-1 beta/IL-1F2 Quantikine HS ELISA Kit, Catalog #: HSLB00D; Human TNF-alpha Quantikine ELISA Kit, Catalog #: DTA00D.</p>
</sec>
<sec id="sec10">
<label>2.8</label>
<title>Quantitative real-time PCR</title>
<p>To determine the levels of expression of the pro-inflammation factors (IL-1&#x03B2;, IL-6, inducible nitric oxide synthase (iNOS), and TNF-&#x03B1;), anti-inflammation factors (IL-10, arginase-1 (ARG), TGF-&#x03B2;, and CD-206), treated retina mRNA was extracted using the Total RNA Extraction Reagent (EZBioscience, Roseville, USA) according to the manufacturer&#x2019;s instructions. Briefly, 1&#x2009;&#x03BC;g total RNA was reverse transcribed to cDNA using a reverse transcription master mix (EZBioscience, Roseville, USA) according to the manufacturer&#x2019;s instructions. Subsequently, qRT-PCR was conducted using an SYBR Green qPCR master mix (ROX2 plus; EZBioscience, Roseville, USA). Cycling parameters were 95&#x00B0;C for 5&#x2009;min, then 40&#x2009;cycles at 95&#x00B0;C for 10&#x2009;s followed by 60&#x00B0;C for 30&#x2009;s. At least three technical replicates were performed for each sample. Relative expression levels were calculated using the 2<sup>&#x2212;&#x0394;&#x0394;Cq</sup> method and are presented as fold-change relative to the glyceraldehyde 3-phosphate dehydrogenase house gene expression. Primers for RT-qPCR are listed in <xref rid="SM1" ref-type="supplementary-material">Supplementary File 2: Table S1</xref>.</p>
</sec>
<sec id="sec11">
<label>2.9</label>
<title>Statistical analysis</title>
<p>Statistical analysis and graph creation were performed with GraphPad Prism 6 (GraphPad Software). Data were presented as the mean&#x2009;&#x00B1;&#x2009;standard error of mean. We used analysis of variance with Tukey&#x2019;s test to adjust for multiple comparisons among more than two groups. Differences were considered statistically significant at <italic>p</italic>-values of &#x003C;0.05.</p>
</sec>
</sec>
<sec sec-type="results" id="sec12">
<label>3</label>
<title>Results</title>
<sec id="sec13">
<label>3.1</label>
<title>Characterization of CEFFE</title>
<p>The CEFFE was obtained from the lipoaspirate of six healthy female volunteers (The mean age was 31&#x2009;years, range 24&#x2013;36&#x2009;years) and was produced, as previously described (<xref ref-type="bibr" rid="ref45">Yu et al., 2018</xref>). Briefly, approximately 7&#x2009;ml of pinkish CEFFE was obtained from 50&#x2009;ml of centrifuged lipoaspirate (collected after the first spin). The total CEFFE protein concentration was 4745.43&#x2009;&#x00B1;&#x2009;751.73&#x2009;&#x03BC;g/ml (<italic>n</italic>&#x2009;=&#x2009;6).</p>
</sec>
<sec id="sec14">
<label>3.2</label>
<title>CEFFE promotes axon regeneration</title>
<p>A single injection of the CEFFE exhibited limited effect on axon regeneration, only a few fibers extended beyond the crush site. The number of regenerated axons was 1,176 at 250&#x2009;&#x03BC;m from the crush site and sharply decreased to 298 at 500&#x2009;&#x03BC;m. At 750 and 1,000&#x2009;&#x03BC;m the number was 37 and 0, respectively. Since injured RGCs underwent several pathological phases until death (<xref ref-type="bibr" rid="ref10">Garcia-Valenzuela et al., 1994</xref>). Within 6&#x2009;h after injury, the number of RGCs did not decrease. On days 3&#x2013;7 post-injury, the rate of RGCs increased gradually, peaking on day 7. So, we adapted our protocol and injected the CEFFE at day 0, day 3 and day 7 after ONC. As a result, robust axon regeneration was observed (<xref ref-type="fig" rid="fig1">Figure 1A</xref>). The number of regenerated axons at different distances in the CEFFE-thrice-injected group was significantly higher than that in the CEFFE-once-injected group, with counts of 2,417, 1745, 970, 569, and 56, respectively. The number of regenerated axons gradually declined, commencing from the lesion site, and the longest regenerated axons reached approximately 1.5&#x2009;mm from the crush site. These results underscore the capacity of repeat CEFFE administration to promote axon regeneration.</p>
<fig position="float" id="fig1">
<label>Figure 1</label>
<caption>
<p>CEFFE promotes axon regeneration. <bold>(A)</bold> Confocal images of optic nerve longitudinal sections showing regenerating fibers labelled with CTB-555. &#x002A; Represents crush site. Scale bar, 100&#x2009;&#x03BC;m. <bold>(B)</bold> Quantification of regenerating axons at different distances distal to the lesion site. &#x002A;&#x002A;&#x002A;<italic>p</italic>&#x2009;&#x003C;&#x2009;0.001, &#x002A;&#x002A;&#x002A;&#x002A;<italic>p</italic>&#x2009;&#x003C;&#x2009;0.0001. Data are presented as means &#x00B1; standard error of mean, <italic>n</italic>&#x2009;=&#x2009;6 mice per group. Two-way analysis of variance with Tukey&#x2019;s test.</p>
</caption>
<graphic xlink:href="fncel-18-1344853-g001.tif"/>
</fig>
</sec>
<sec id="sec15">
<label>3.3</label>
<title>CEFFE is better than a single neurotrophic factor for axon regeneration</title>
<p>Both CEFFE and CNTF were intravitreally injected on days 0, 3, and 7 post-crush (<xref ref-type="fig" rid="fig2">Figure 2</xref>). When utilizing CNTF (5&#x2009;&#x03BC;g/&#x03BC;l), it was observed to induce axon regeneration to a certain extent. The counts of regenerated axons in the CNTF-treated group were 1,060, 593, 263, 88, and 2 at different distances from the crush site, respectively. However, under identical concentration conditions (5&#x2009;&#x03BC;g/&#x03BC;l), the number of regenerated axons in the CEFFE-treated group significantly exceeded that of the CNTF-treated group. Specifically, the counts were 2,268, 1,264, 660, 331, and 41 in the CEFFE-treated group. These results, as depicted in <xref ref-type="fig" rid="fig2">Figure 2</xref>, clearly demonstrate that the impact of the CEFFE on axon regeneration surpassed that of a single trophic factor.</p>
<fig position="float" id="fig2">
<label>Figure 2</label>
<caption>
<p>CEFFE induces more robust axon regeneration than CNTF. <bold>(A)</bold> Confocal images of optic nerve sections showing regenerating axons labeled by CTB-555 2&#x2009;weeks after optic nerve injury from PBS, CNTF- (5&#x2009;&#x03BC;g/&#x03BC;l), and CEFFE-treated (5&#x2009;&#x03BC;g/&#x03BC;l) eyes. &#x002A; Represents crush site. Scale bar, 100&#x2009;&#x03BC;m. <bold>(B)</bold> Quantification of regenerating axons at different distances distal to the lesion site. Data are presented as mean&#x2009;&#x00B1;&#x2009;standard error of mean, <italic>n</italic>&#x2009;=&#x2009;6 mice per group. &#x002A;<italic>p</italic>&#x003C;0.05, &#x002A;&#x002A;&#x002A;<italic>p</italic>&#x003C;0.001, &#x002A;&#x002A;&#x002A;&#x002A;<italic>p</italic>&#x2009;&#x003C;&#x2009;0.0001, Two-way analysis of variance with Tukey&#x2019;s test.</p>
</caption>
<graphic xlink:href="fncel-18-1344853-g002.tif"/>
</fig>
</sec>
<sec id="sec16">
<label>3.4</label>
<title>CEFFE promotes RGCs survival</title>
<p>In addition to promoting axon regeneration, we found that the CEFFE had a significant protective effect on RGCs survival. In contrast to 14.37% of RGCs surviving rate in the PBS-treated group, approximately 54.5% of RGCs survived after the CEFFE injection (see <xref ref-type="fig" rid="fig3">Figure 3</xref>), which was significantly higher than CNTF-treated group (36.61%). These results indicate the neuroprotective effects of CEFFE.</p>
<fig position="float" id="fig3">
<label>Figure 3</label>
<caption>
<p>CEFFE promotes RGCs survival after optic nerve crush. <bold>(A)</bold> Confocal images of retinal whole-mounts show surviving Tuj1+ RGCs at 2&#x2009;weeks after optic nerve injury. Scale bar, 25&#x2009;mm. <bold>(B)</bold> Quantification of RGCs survival at 2&#x2009;weeks after injury, expressed as a percentage of total Tuj1+ RGCs in the uninjured retina. Data are presented as the mean&#x2009;&#x00B1;&#x2009;standard error of mean, <italic>n</italic>&#x2009;=&#x2009;3 mice per group. &#x002A;&#x002A;<italic>p</italic>&#x2009;&#x003C;&#x2009;0.01, One-way analysis of variance with Tukey&#x2019;s test.</p>
</caption>
<graphic xlink:href="fncel-18-1344853-g003.tif"/>
</fig>
</sec>
<sec id="sec17">
<label>3.5</label>
<title>CEFFE comprises multiple factors related to axongenesis and neurogenesis</title>
<p>To gain a better understanding of CEFFE, we analyzed its components. We have previously reported detailed bioinformatics information (including total protein levels and GO classification) of CEFFE (<xref ref-type="bibr" rid="ref45">Yu et al., 2018</xref>). In this study, delved deeper into the proteomic data, specifically identifying and categorizing proteins related to neurogenesis or axongenesis. We found 146 proteins involved in axongenesis or neurogenesis and sorted out according to subcellular localization (see <xref rid="SM1" ref-type="supplementary-material">Supplementary File 1: Table S1</xref>). Among which, Signal transducer and activator of transcription 3 (STAT3), Ras-related C3 botulinum toxin substrate 1 (RAC1), and cell division control protein 42 homolog (CDC42), have been shown to regulate axon regeneration (<xref ref-type="bibr" rid="ref26">Lorenzetto et al., 2013</xref>; <xref ref-type="bibr" rid="ref35">Namekata et al., 2014</xref>; <xref ref-type="bibr" rid="ref31">Mehta et al., 2016</xref>). Since the regeneration of damaged axons share some similar processes as does axon growth during development (<xref ref-type="bibr" rid="ref12">He and Jin, 2016</xref>; <xref ref-type="bibr" rid="ref28">Mahar and Cavalli, 2018</xref>), we speculated that these factors in CEFFE may contribute to axon regeneration and provide neuroprotection.</p>
</sec>
<sec id="sec18">
<label>3.6</label>
<title>CEFFE promotes axon regeneration and RGCs survival from diverse aspects</title>
<p>To further explain the mechanism underlying the regeneration phenomenon, we conducted experiments aimed at monitoring changes within retina. Firstly, we perform ELISA experiment to examine the expression of inflammation factors in CEFFE. Studies have demonstrated that elevated levels of pro-inflammatory cytokines such as IL-1&#x03B2; and TNF&#x03B1; are neurotoxic and closely associated with substantial axonal damage (<xref ref-type="bibr" rid="ref18">Kitaoka et al., 2017</xref>; <xref ref-type="bibr" rid="ref33">Mou et al., 2021</xref>). Conversely, the anti-inflammatory factors TGF&#x03B2;-1 and IL-4 have been suggested to promote axonal growth during the inflammatory response (<xref ref-type="bibr" rid="ref1">Andries et al., 2020</xref>). The mean level and variation of pro-inflammatory (TNF&#x03B1; and IL-1&#x03B2;) and anti-inflammatory factors (TGF&#x03B2;-1and IL-4) were summarized in <xref ref-type="table" rid="tab1">Table 1</xref>.</p>
<table-wrap position="float" id="tab1">
<label>Table 1</label>
<caption>
<p>Mean inflammation factors concentration in CEFFE (<italic>n</italic> =&#x2009;6, pg./ml).</p>
</caption>
<table frame="hsides" rules="groups">
<thead>
<tr>
<th/>
<th align="left" valign="top">TGF&#x03B2;-1</th>
<th align="left" valign="top">IL-4</th>
<th align="left" valign="top">TNF&#x03B1;</th>
<th align="left" valign="top">IL-1&#x03B2;</th>
</tr>
</thead>
<tbody>
<tr>
<td align="left" valign="bottom">Mean</td>
<td align="center" valign="top">492.01</td>
<td align="center" valign="top">15.70</td>
<td align="center" valign="top">48.9</td>
<td align="center" valign="top">0.9</td>
</tr>
<tr>
<td align="left" valign="bottom">SEM</td>
<td align="center" valign="top">195.22</td>
<td align="center" valign="top">6.55</td>
<td align="center" valign="top">13.49</td>
<td align="center" valign="top">1.37</td>
</tr>
</tbody>
</table>
</table-wrap>
<p>Considering the well-documented role of mTOR in regulating anabolic metabolism and its potential significance in supporting axon regenerative growth in injured neurons (<xref ref-type="bibr" rid="ref37">Park et al., 2008</xref>; <xref ref-type="bibr" rid="ref27">Lu et al., 2014</xref>; <xref ref-type="bibr" rid="ref7">Curcio and Bradke, 2018</xref>), we investigated the expression of mTOR. Conversely, we also examined the expression of ROCK2, a known inhibitor of axon regeneration (<xref ref-type="bibr" rid="ref30">McKerracher et al., 2012</xref>). and cleaved-calpain as an indicator of apoptosis in the retina. As shown in <xref ref-type="fig" rid="fig4">Figure 4</xref>, compared with PBS treated group, the expression of mTOR was about 1.6-fold in CEFFE-treated group. While the expression of ROCK2, and cleaved-calpain reduced to 0.5 and 0.2-fold, respectively.</p>
<fig position="float" id="fig4">
<label>Figure 4</label>
<caption>
<p>CEFFE modulates apoptosis and metabolism in retina. <bold>(A)</bold> Western blots detected the expression of mTOR, ROCK2 and cleaved-calpain in retinal lysates from PBS-treated and CEFFE-treated groups. <bold>(B)</bold> Quantification of band intensities normalized to &#x03B2;-actin as a loading control and relative to that of PBS are depicted (bars represent mean&#x2009;&#x00B1;&#x2009;standard error of mean, <italic>n</italic>&#x2009;=&#x2009;3 mice per group). &#x002A;&#x002A;&#x002A;&#x002A;<italic>p</italic>&#x2009;&#x003C;&#x2009;0.0001, Two-way analysis of variance with Tukey&#x2019;s test.</p>
</caption>
<graphic xlink:href="fncel-18-1344853-g004.tif"/>
</fig>
<p>At last, our observations indicated that CEFFE exhibited an inhibitory effect on microglia activation (see <xref ref-type="fig" rid="fig5">Figures 5A</xref>,<xref ref-type="fig" rid="fig5">B</xref>). The use of CD11b to label activated microglia revealed that the total number of CD11b&#x2009;+&#x2009;cells was 85.2&#x2009;&#x00B1;&#x2009;4.4 in the PBS-treated group, but notably reduced to 66.2&#x2009;&#x00B1;&#x2009;3.59 in the CEFFE-treated group within the entire retina section. This decline in the number of activated microglia was significant. Additionally, in GCL, when compared to the control group (44.8&#x2009;&#x00B1;&#x2009;3.76), the number of activated microglia in the CEFFE-treated group was significantly reduced to 29.6&#x2009;&#x00B1;&#x2009;2.71. To further explore the potential role of CEFFE in resolving inflammation, we conducted RT-qPCR for proinflammatory and anti-inflammatory factors in retina (see <xref ref-type="fig" rid="fig5">Figures 5C</xref>,<xref ref-type="fig" rid="fig5">D</xref>). Our results showed that the mRNA expression of anti-inflammatory factors (ARG, IL-10, CD-206 and TGF-&#x03B2;) were remarkably elevated. In contrast, except for TNF-&#x03B1;, the changes of pro-inflammatory factors (IL-1&#x03B2;, IL-6 and iNOS) were not statistically significant. These findings collectively provide insights into the multifaceted mechanisms underlying CEFFE&#x2019;s regenerative and neuroprotective effects.</p>
<fig position="float" id="fig5">
<label>Figure 5</label>
<caption>
<p>CEFFE attenuates microglia activation and modulate inflammation factors in retina. <bold>(A)</bold> Representative confocal pictures of sections showing CD11b-positive microglia (green) in retina. (GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer plexiform layer). Scale bar, 50&#x2009;&#x03BC;m. <bold>(B)</bold> Quantification of CD11b-positive cells in GCL and entire retina. <bold>(C)</bold> Quantification of ARG, IL-10, CD206, and TGF-&#x03B2; mRNA expression by RT-qPCR. <bold>(D)</bold> Quantification of IL-1&#x03B2;, IL-6, iNOS, and TNF-&#x03B1; mRNA expression by RT-qPCR. One-way ANOVA and Tukey&#x2019;s multiple comparisons test. &#x002A;<italic>p</italic>&#x2009;&#x003C;&#x2009;0.05, &#x002A;&#x002A;<italic>p</italic>&#x2009;&#x003C;&#x2009;0.01, &#x002A;&#x002A;&#x002A;<italic>p</italic>&#x2009;&#x003C;&#x2009;0.001, &#x002A;&#x002A;&#x002A;&#x002A;<italic>p</italic>&#x2009;&#x003C;&#x2009;0.0001 (<italic>n</italic>&#x2009;=&#x2009;5 mice per group).</p>
</caption>
<graphic xlink:href="fncel-18-1344853-g005.tif"/>
</fig>
</sec>
</sec>
<sec sec-type="discussion" id="sec19">
<label>4</label>
<title>Discussion</title>
<p>The secretome from the ADSCs has been used in pre-clinical studies of traumatic brain injury (<xref ref-type="bibr" rid="ref41">Tajiri et al., 2014</xref>; <xref ref-type="bibr" rid="ref14">Jha et al., 2018</xref>; <xref ref-type="bibr" rid="ref38">Patel et al., 2018</xref>). Moreover, in some studies, the secretome from the ADSCs emerged as superior to that from the BMSCs in the treatment of neurodegenerative diseases (<xref ref-type="bibr" rid="ref17">Katsuda et al., 2013</xref>; <xref ref-type="bibr" rid="ref19">Lee et al., 2016</xref>). Researcher believed that its neuroprotective role is primarily mediated through paracrine mechanism, and found effective axon regeneration needs a synergistic interplay among various neurotrophic factors (<xref ref-type="bibr" rid="ref13">Huang and Reichardt, 2001</xref>; <xref ref-type="bibr" rid="ref25">Logan et al., 2006</xref>; <xref ref-type="bibr" rid="ref20">Leibinger et al., 2016</xref>). CEFFE is a mixture of diverse proteins, including trophic factors, inflammation factors, and chemokines. The findings depicted in <xref ref-type="fig" rid="fig1">Figure 1</xref> corroborate the efficacy of CEFFE in promoting axon regeneration. To ascertain the superior performance of CEFFE in comparison to a single trophic factor, we employed CNTF, one of the most typical trophic factors involved in axon regeneration (<xref ref-type="bibr" rid="ref34">M&#x00FC;ller et al., 2007</xref>), as a control. At the same condition, CEFFE showed more robust ability in axon regeneration compared with CNTF. It is plausible that a convergence of these factors acted in concert to activate signaling pathways vital to axon regeneration. For instance, both HGF and BDNF possess the capability to stimulate the mTOR pathway (<xref ref-type="bibr" rid="ref40">Segarra et al., 2006</xref>; <xref ref-type="bibr" rid="ref23">Lima Giacobbo et al., 2019</xref>). As the results shown in <xref ref-type="fig" rid="fig4">Figure 4</xref>, CEFFE increased mTOR expression and decreased ROCK2 expression.</p>
<p>Promoting neuronal survival and axon regeneration are two basic strategies to repair injuries in mature CNS. While modulation of intrinsic regulators can markedly alter the regenerative potential of neurons, singular gene manipulation can sometimes introduce incongruence in axon regeneration or neuronal survival. This has been exemplified in the cases of Armcx and Sox11 (<xref ref-type="bibr" rid="ref2">Belin et al., 2014</xref>; <xref ref-type="bibr" rid="ref22">Li et al., 2018</xref>). The evidence presented in <xref ref-type="fig" rid="fig3">Figure 3</xref> demonstrates that CEFFE effectively safeguards RGCs from apoptosis. Moreover, the data in <xref ref-type="fig" rid="fig4">Figure 4</xref> indicates a reduction in the expression of cleaved-calpain, a molecule associated with RGCs apoptosis (<xref ref-type="bibr" rid="ref6">Cheng et al., 2018</xref>). In conjunction with the findings in <xref ref-type="fig" rid="fig1">Figure 1</xref>, our research posits that employing CEFFE can concurrently support both axon regeneration and the survival of RGCs.</p>
<p>Unlike the secretome of the ADSCs, CEFFE comprise both bioactive extracellular and intracellular components, as shown by our proteomics findings (see <xref rid="SM1" ref-type="supplementary-material">Supplementary File 1: Table S1</xref>). That may explain why the concentration of some trophic factors in CEFFE is much higher than that in the secretome from stem cells (<xref ref-type="bibr" rid="ref39">Ribeiro et al., 2012</xref>; <xref ref-type="bibr" rid="ref16">Johnson et al., 2014</xref>; <xref ref-type="bibr" rid="ref29">Martins et al., 2017</xref>). For example, the concentration of the BDNF in CEFFE was approximately 1860.99&#x2009;pg./ml, while that reported in other studies was approximately 13.22 or 37.03&#x2009;pg./ml (<xref ref-type="bibr" rid="ref16">Johnson et al., 2014</xref>; <xref ref-type="bibr" rid="ref29">Martins et al., 2017</xref>; <xref ref-type="bibr" rid="ref45">Yu et al., 2018</xref>). In addition to trophic factors, CEFFE exhibits considerably higher concentrations of inflammatory factors, as detailed in <xref ref-type="table" rid="tab1">Table 1</xref>, in contrast to the mesenchymal stem cell secretome (<xref ref-type="bibr" rid="ref16">Johnson et al., 2014</xref>). Intraocular inflammation promotes RGCs survival and axon regeneration (<xref ref-type="bibr" rid="ref21">Leon et al., 2000</xref>; <xref ref-type="bibr" rid="ref3">Benowitz and Popovich, 2011</xref>). However, persistent chronic inflammation will ultimately lead to RGCs death and axon degeneration. CEFFE demonstrates a relatively rapid <italic>in vivo</italic> degradation profile, and the anti-inflammatory factors (TGF&#x03B2;-1 and IL-4) in CEFFE are more dominant in content than the pro- inflammatory factors (TNF&#x03B1; and IL-1&#x03B2;). This observation suggests that repeated, short-acting inflammatory stimulation elicited by CEFFE injections may explain the possible therapeutic mechanism of CEFFE from another perspective.</p>
<p>In another aspect, microglia, which serve as long-lived resident macrophages within the retinal parenchyma, undergo progressive activation subsequent to optic nerve injury. This activation process culminates in the production of pro-inflammatory cytokines and the generation of reactive oxygen species, both of which exhibit neurotoxic effects upon RGCs. Studies have proven that diminishing microglial activation, as observed in glaucoma and other CNS models, reduces neuronal cell death and mitigates neuroinflammation (<xref ref-type="bibr" rid="ref5">Bosco et al., 2008</xref>; <xref ref-type="bibr" rid="ref42">Takeda et al., 2018</xref>). Owing to its lack of immunogenicity and antioxidant effects, CEFFE is promising for relieve chronic inflammation. Our results showed that CEFFE could attenuate microglia activation after optic nerve crush (see <xref ref-type="fig" rid="fig5">Figure 5</xref>). The changes of anti and pro-inflammation factors indicated that after treated with CEFFE the microglia in retina presented an anti-inflammation state. The controlled microglia activation caused by CEFFE may contribute to axon regeneration and RGCs survival furtherly.</p>
<p>This study is not without its limitations. Although our results suggest that CEFFE operates as a cohesive unit, we must acknowledge the intricacies and vast network of factors involved. As a consequence, pinpointing the precise combination of factors within CEFFE that influence axon regeneration and the survival of RGCs has remained elusive. It is plausible that the effects of CEFFE encompass several intricate mechanisms, and isolating the principal mode of action proves challenging. To provide more comprehensive insights into the therapeutic potential of CEFFE, further investigations are warranted. Future studies should seek to delineate the temporal dynamics of CEFFE&#x2019;s action <italic>in vivo</italic> and endeavor to optimize the selection of its active constituents. In summary, our study indicates that CEFFE can enhance axon regeneration and improve RGCs survival after optic nerve crush. As an allogeneic biological agent, CEFFE has the advantage of no cell component, no immunogenicity, etc. Therefore, it represents a potential therapeutic avenue for CNS injury.</p>
</sec>
<sec sec-type="data-availability" id="sec20">
<title>Data availability statement</title>
<p>The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.</p>
</sec>
<sec sec-type="ethics-statement" id="sec21">
<title>Ethics statement</title>
<p>The studies involving humans were approved by Institutional Ethics Committee of Shanghai Ninth People&#x2019;s Hospital, Shanghai Jiaotong University School of Medicine. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. The animal study was approved by Institutional Ethics Committee of Shanghai Ninth People&#x2019;s Hospital, Shanghai Jiaotong University School of Medicine. The study was conducted in accordance with the local legislation and institutional requirements.</p>
</sec>
<sec sec-type="author-contributions" id="sec22">
<title>Author contributions</title>
<p>YS: Data curation, Formal analysis, Methodology, Writing &#x2013; original draft. DC: Data curation, Formal analysis, Methodology, Writing &#x2013; original draft. TD: Software, Writing &#x2013; review &#x0026; editing. ZY: Software, Writing &#x2013; review &#x0026; editing. HX: Writing &#x2013; review &#x0026; editing. XW: Conceptualization, Writing &#x2013; original draft. WZ: Conceptualization, Funding acquisition, Writing &#x2013; review &#x0026; editing.</p>
</sec>
</body>
<back>
<sec sec-type="funding-information" id="sec23">
<title>Funding</title>
<p>The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This work was supported by the Shanghai Collaborative Innovation Program on Regenerative Medicine and Stem Cell Research (2019CXJQ01), National Natural Science Foundation of China (Grant No. 82002060), Construction Fund of Medical Key Disciplines of Hangzhou (0020200044), Medical and Health Science and Technology Program of Hangzhou (A20220646).</p>
</sec>
<ack>
<p>Pre-print version of this paper was published in Research Square. DOI: <ext-link xlink:href="https://doi.org/10.21203/rs.3.rs-655351/v1" ext-link-type="uri">10.21203/rs.3.rs-655351/v1</ext-link>.</p>
</ack>
<sec sec-type="COI-statement" id="sec24">
<title>Conflict of interest</title>
<p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>
</sec>
<sec id="sec100" sec-type="disclaimer">
<title>Publisher&#x2019;s note</title>
<p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p>
</sec>
<sec sec-type="supplementary-material" id="sec25">
<title>Supplementary material</title>
<p>The Supplementary material for this article can be found online at: <ext-link xlink:href="https://www.frontiersin.org/articles/10.3389/fncel.2024.1344853/full#supplementary-material" ext-link-type="uri">https://www.frontiersin.org/articles/10.3389/fncel.2024.1344853/full#supplementary-material</ext-link></p>
<supplementary-material xlink:href="Image_1.JPEG" id="SM1" mimetype="image/jpeg" xmlns:xlink="http://www.w3.org/1999/xlink"/>
<supplementary-material xlink:href="Table_1.DOCX" id="SM2" mimetype="application/vnd.openxmlformats-officedocument.wordprocessingml.document" xmlns:xlink="http://www.w3.org/1999/xlink"/>
<supplementary-material xlink:href="Table_2.DOCX" id="SM3" mimetype="application/vnd.openxmlformats-officedocument.wordprocessingml.document" xmlns:xlink="http://www.w3.org/1999/xlink"/>
</sec>
<fn-group>
<fn id="fn0001">
<p><sup>1</sup><ext-link xlink:href="http://www.ebi.ac.uk/GOA/" ext-link-type="uri">http://www.ebi.ac.uk/GOA/</ext-link>
</p>
</fn>
<fn id="fn0002">
<p><sup>2</sup><ext-link xlink:href="http://www.ebi.ac.uk/interpro/" ext-link-type="uri">http://www.ebi.ac.uk/interpro/</ext-link>
</p>
</fn>
<fn id="fn0003">
<p><sup>3</sup><ext-link xlink:href="http://geneontology.org/" ext-link-type="uri">http://geneontology.org/</ext-link>
</p>
</fn>
</fn-group>
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