# Brain inflammation and cognitive decline induced by spinal cord injury can be reversed by spinal cord cell transplants

Quentin Delarue, Amandine Robac, Fannie Semprez, Célia Duclos, Baptiste

Pileyre, Pauline Neveu, Clémence Raimond, Gaëtan Riou, Inès Ziane, Nicolas

Guérout

► To cite this version:

Quentin Delarue, Amandine Robac, Fannie Semprez, Célia Duclos, Baptiste Pileyre, et al.. Brain inflammation and cognitive decline induced by spinal cord injury can be reversed by spinal cord cell transplants. Brain, Behavior, and Immunity, 2025, 125, pp.388-397. 10.1016/j.bbi.2025.01.014. hal-05461264

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Short Communication

# Brain inflammation and cognitive decline induced by spinal cord injury can be reversed by spinal cord cell transplants

Quentin Delarue<sup>a,b,*</sup>, Amandine Robac<sup>a</sup>, Fannie Semprez<sup>b</sup>, Célia Duclos<sup>a</sup>, Baptiste Pileyre<sup>c,d</sup>, Pauline Neveu<sup>b</sup>, Clémence Raimond<sup>b</sup>, Gaëtan Riou<sup>c</sup>, Inès Ziane<sup>b</sup>, Nicolas Guérout<sup>b,*</sup>

<figure><img src="image_2.png" /></figure>

ARTICLE INFO

Keywords:

Spinal cord injury

Cell transplantation

Brain inflammation

Cognitive impairment

Neurogenesis

ABSTRACT

Spinal cord injuries (SCI) impact between 250,000 and 500,000 people worldwide annually, often resulting from road accidents or falls. These injuries frequently lead to lasting disabilities, with the severity depending on the injury's extent and location. Emerging research also links SCI to cognitive impairments due to brain inflammation. From a treatment perspective, various approaches, including cell therapy, have been investigated. One common mechanism across cellular transplant models is the modulation of inflammation at the injury site, though it remains uncertain if these effects extend to the brain. To explore this, we induced SCI in wild-type mice and treated them with either olfactory ensheathing cells or mesenchymal stem cells. Our findings reveal that both cell types can reverse SCI-induced cognitive deficits, reduce brain inflammation, and increase hippocampal neuronal density. This study is the first, to our knowledge, to demonstrate that cells transplanted into the spinal cord can influence brain inflammation and mitigate injury-induced effects on brain functions. These results highlight the intricate relationship between the spinal cord and brain in both health and disease.

# 1. Introduction

Spinal cord injuries (SCI) are most commonly caused by trauma from road accidents or falls. They predominantly affect men, with the highest incidence among young adults and those over 60 (Golestani et al., 2022; Liu et al., 2023). Depending on their severity and location, SCI result in the loss of motor, sensory, and autonomic functions, and currently, there is no cure.

The pathophysiology of SCI has been studied for many years and is well documented (Anjum et al., 2020). More recently, research has focused on the effects of SCI on the brain, showing that they also cause inflammation in brain parenchyma. This inflammation is responsible for a reduction in hippocampal neurogenesis, leading to cognitive deficits in human and mice (Craig et al., 2017; Li et al., 2020; Maldonado-Bouchard et al., 2016). Therapeutic approaches most frequently studied to repair damaged spinal cords include cell transplantation (Assinck et al., 2017) and, more recently, electrical (Wagner et al., 2018) and magnetic neuromodulation techniques (Chalfouh et al., 2020; Robac et al., 2021). Historically, various cell types have been investigated in

the context of SCI, including differentiated cells like bulbar olfactory ensheathing cells (bOECs) and stem cells such as mesenchymal stem cells (MSCs). For the past thirty years, bOECs have been explored in preclinical animal studies and clinical human trials in the context of SCI (Gómez et al., 2018). It has been shown that these cells can modulate spinal cord scar, promote the regrowth or survival of motor fibers, and enable functional recovery without integrating into the spinal tissue (Delarue et al., 2021; Wang et al., 2022). Interestingly, a growing body of research indicates that the primary role of bOECs after transplantation is to exert an immunomodulatory effect on microglia and macrophages present at the lesion site (Khankan et al., 2016; Zhang et al., 2021). Regarding MSCs, these non-neural cells have also shown great interest in tissue regeneration after SCI in animal models and humans. They stimulate spinal cord tissue regeneration, axonal regrowth (Chen et al., 2022; Tien et al., 2019) and like bOECs, provide an immunomodulatory effect (Han et al., 2015; Nakajima et al., 2012).

Based on this knowledge, we asked ourselves: just as inflammation in the spinal cord can lead to disruptions in the brain following injury, can the immunomodulatory effects of bOECs or MSCs transplanted into the

<footnote><sup>a</sup> Univ Rouen Normandie, Normandie Univ, GRHVN UR 3830, F-76000 Rouen, France</footnote>

<footnote><sup>b</sup> Université Paris Cité, CNRS UMR8003, Saints-Pères Paris Institute for Neurosciences, F-75006 Paris, France</footnote>

<footnote><sup>c</sup> Normandie Univ, UNIROUEN, Inserm, U1234, FOCIS Center of Excellence, Pan'THER, F-76000 Rouen, France</footnote>

<footnote><sup>d</sup> Centre Henri Becquerel, Department of Pharmacy, F-76000 Rouen, France</footnote>

<footnote>* Corresponding authors: at: Univ Rouen Normandie, Normandie Univ, GRHVN UR 3830, F-76000 Rouen, France (Quentin Delarue). E-mail addresses: quentindelarue.rd@gmail.com (Q. Delarue), nicolas.guerout@u-paris.fr (N. Guérout).</footnote>
spinal cord after SCI induce beneficial effects on brain tissue and help restore in the recovery of cognitive performance?

Therefore, our study represents the first investigation into the impact of spinal cord transplantation of bOECs or MSCs following SCI on brain inflammation and cognitive functions.

## 2. Materials and methods

### 2.1. Animal care and use statement

The animal protocol was designed to minimize pain and discomfort for animals. All experimental procedures complied with the European Community guiding principles on the care and use of animals (86/609/CEE; Official Journal of the European Communities No. L358; December 18, 1986), the French Decree No. 97/748 of October 19, 1987 (Journal Officiel de la République Française; October 20, 1987), and the recommendations of the Cenomexa Ethics Committee (#26905).

### 2.2. Animals

Mice of both sexes were group-housed (two to five mice per cage, with genders separated) in secure conventional rodent facilities, maintained on a 12-hour light/dark cycle with constant access to food and water. A total of 81 wild-type C57BL/6 mice were used in this study for cell preparation and group allocation. All experiments were conducted on adult mice aged 8-12 weeks, with an average weight of 20 g for females and 25 g for males. Experimental groups were formed with an equal sex ratio (50:50). Our study consisted of four main experimental groups (n = 8-10 per group):

Non-injured control group: mice without SCI used to establish baselines in histological and functional experiments (Figs. 1a and 2a).

SCI control group: mice underwent SCI without transplantation (Fig. 1a and 2a).

SCI + bOECs group: mice underwent SCI and received primary culture of bOECs (Fig. 1a).

SCI + MSCs group: mice underwent SCI and received MSCs (Fig. 2a).

### 2.3. Primary olfactory bulb culture and cell transplant preparation

Olfactory bulb (OB) primary cultures were prepared as previously described (Delarue et al., 2019; Guérout et al., 2010). Briefly, mice were anesthetized with 2 % isoflurane (Iso-Vet, Osalia, Paris, France) and then euthanized by decapitation. OBs were extracted from the brain and placed in cold PBS. The tissue was incubated in 0.25 % trypsin (2.5 % Trypsin 10X, 15090-046, Thermo Fisher) and mechanically dissociated. Once dissociated, cells were cultured in T25 flasks with 5 mL of DF-10S medium (DMEM + GlutaMAX, 0.5 % Penicillin/Streptomycin, and 10 % heat-inactivated fetal bovine serum). Three weeks after plating, cultures were trypsinized and cells were counted to prepare a cell suspension at 25,000 cells/µL in DF-10S for transplantation.

### 2.4. Mesenchymal stem cell culture and cell transplant preparation

MSCs cultures were prepared from adipose tissue. Briefly, mice were anesthetized with 2 % isoflurane (Iso-Vet, Osalia, Paris, France) and then euthanized by decapitation. After removal of the lymph node, inguinal white adipose tissue was collected and placed in cold PBS. The tissue was minced with a syringe and dissociated using NB5 collagenase (Nordmak, N0002778) on an orbital shaker for 1 h. Once dissociated, cells were cultured in T75 flasks with 15 mL of medium (DMEM + GlutaMAX, 1 % Penicillin/Streptomycin, and 20 % heat-inactivated fetal bovine serum). One week after plating, cultures were trypsinized and replated in T75 flasks. Cultures were passaged three times to obtain purified MSCs. After three passages, cells were counted, and a cell suspension was prepared at 50,000 cells/µL in culture medium for transplantation.

### 2.5. Flow cytometry and bOECs characterization

Cells were characterized by flow cytometry three weeks after plating. Primary bOECs were adjusted to a density of $2 \times 10^5$ cells/mL in PBS with 0.5 % bovine serum albumin (BSA) solution. TruStain FcX™ PLUS (BioLegend) was used to block non-specific binding. Cell populations were identified using primary antibodies: anti-p75 nerve growth factor receptor (p75, Abcam, ab8874, RRID) and rat anti-platelet-derived growth factor β (PDGFrβ, Abcam, ab91066, RRID). OECs were identified as p75-positive and PDGFrβ-negative cells, while stromal cells were identified as PDGFrβ-positive and p75-negative cells. p75 and PDGFrβ primary antibodies were revealed with anti-rabbit phycoerythrin (PE, poly4064, BioLegend, 406408, RRID) and anti-rat Alexa Fluor 488 (AF488, MRG2b-85, BioLegend, RRID) fluorochrome-conjugated secondary antibodies, respectively. All analyses were performed on a BD FortessaTM cytometer (Becton Dickinson) and post-analyzed using FlowJo software (version 10.3; FlowJo LLC).

### 2.6. Flow cytometry and MSCs characterization

Cells were characterized by flow cytometry after three passages. MSCs were adjusted to a density of $2 \times 10^5$ cells/mL in PBS with 0.5 % BSA solution. TruStain FcX™ PLUS (BioLegend) was used to block non-specific binding. MSCs were characterized as CD105-positive, CD31-negative, and CD34-negative cells. In contrast, endothelial cells were defined as CD105-negative, CD31-positive, and CD34-negative, while hematopoietic cells were identified as CD105-negative, CD31-negative, and CD34-positive. Cells were labeled with the following antibodies: anti-endoglin conjugated with peridinin-chlorophyll-protein complex-cyanine5.5 (CD105-PerCP-Cy5.5, Sony, 1202080), anti-PECAM1 conjugated with phycoerythrin (CD31-PE, Sony, 1112040), and anti-mucosialin conjugated with allophycocyanin (CD34-APC, Sony, 1243060). All analyses were performed on a BD FortessaTM cytometer and post-analyzed using FlowJo software.

### 2.7. Surgical procedure and cell transplantation

SCI was performed as previously described (Delarue et al., 2019) at the T9-T10 level under anesthesia (2 % isoflurane). After a dorsal incision and laminectomy at the T7 vertebra, an incomplete transection of the spinal cord was performed to preserve the lower part of the ventral horns while completely injuring the central part and the dorsal horns. The SCI was made using a 25-gauge needle. Immediately following the lesion, bOECs or MSCs were transplanted. Both bOECs and MSCs were transplanted using a micromanipulator arm fixed to a stereotaxic frame (World Precision Instruments). Cells were injected with a 1 mm sterile glass capillary needle attached to the micromanipulator. Injections were delivered at a 1 mm depth, 1.5 mm from the midline, and 5 mm rostrally and caudally from the lesion site. Two injections of 2 µL each, containing 25,000 cells/µL of bOECs or 50,000 cells/µL of MSCs, were administered. Each injection was carefully delivered over 1 min. After surgery, back muscles and skin were sutured, and mice were monitored daily. None of the animals showed signs of skin lesions, infection, or autopathy throughout the study.

### 2.8. Novel object recognition (NOR)

NOR experiments allow for the assessment of memory capacity and exploratory behavior. Briefly, the day before the test, mice were individually placed twice, at the beginning and end of the day, in a 35 × 35 cm open field for 5 min each time for habituation. The next day, mice were placed in the open field with two identical objects for 10 min (training session). After 2 h, object recognition was assessed by replacing one of the familiar objects with a novel object that differed in color, shape, size, and texture. Mice were then individually placed in the same open field for 10 min, and the time spent exploring the familiar and
a.

A - No SCI group

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B - SCI group

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C - SCI + bOECs group

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Dissociation and culture

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b.

Percentage of marker positive cells (%)

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<table><thead><tr><th></th><th>Mean</th><th>SD</th></tr></thead><tbody><tr><td>Stromal<br>Cells - PDGFrb</td><td>63,8%</td><td>6,9%</td></tr><tr><td>Olfac. Ensheat.<br>Cells - p75 high</td><td>28,5%</td><td>5,5%</td></tr><tr><td>Olfac. Ensheat.<br>Cells - p75 low</td><td>5,9%</td><td>1,1%</td></tr></tbody></table>

c.

Training Session

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Testing Session

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d.

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e.

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f.

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j.

14 days post-SCI

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g.

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h.

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n.

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s.

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t.

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(caption on next page)
**Fig. 1.** Transplantation of bOECs reduces hippocampal inflammation, increases neurogenesis and corrects the cognitive deficit induced by SCI. a. Schematic representation of the three experimental groups: A – No SCI group, B – SCI group, and C – SCI + bOECs group. b. Cytometric characterization of bOECs primary culture. Anti-PDGFrβ and anti-p75 antibodies were used to identify stromal cells and OECs, respectively. c-i. NOR experiments were conducted 64 days after SCI. c. Schematic representation of the NOR experiment. After a training session with two identical objects, one object was replaced by a new object different from the first. During the testing session, the frequency and duration of head orientation towards the new object were measured. d. Frequency of head orientation towards the familiar object at 64 days post-SCI. e. Frequency of head orientation towards the new object at 64 days post-SCI. f. Cumulative frequency of head orientation towards both objects at 64 days post-SCI. g. Duration of head orientation towards the familiar object at 64 days post-SCI. h. Duration of head orientation towards the new object at 64 days post-SCI. i. Cumulative duration of head orientation towards both objects at 64 days post-SCI. j-t. Immunohistology experiments. j and k. Quantification of immature neurons (DCX+) in the DG 14 days post-SCI. j. Quantification of DCX+ neuron density in the DG 14 days post-SCI. k. Representative images of sagittal DG sections from SCI (k1 and k1') and SCI + bOECs (k2 and k2') groups 14 days post-SCI, stained with anti-DCX antibody. k1 and k2: Scale bar = 100 µm; k1' and k2': Scale bar = 50 µm. l and m. Quantification of mature neuron (NeuN+) density in the DG, CA3, CA2, and CA1 64 days post-SCI. l. Representative images of hippocampal sagittal sections from the SCI (l1) and SCI + bOECs (l2) groups, stained with anti-NeuN antibody. l1 and l2: Scale bar = 100 µm; DG, CA1, CA2, and CA3: Scale bar = 50 µm. m. Quantification of NeuN+ neuron density in the hippocampus 64 days post-SCI. n. Quantification of astrocytes density (GFAP+) in the hippocampus and 64 days post-SCI. o. Representative images of hippocampal sagittal sections from SCI + bOECs, stained with anti-GFAP antibodies. o1 and o2: Scale bar = 100 µm; o1' and o2': Scale bar = 50 µm. p-t. Analysis of microglial cell in the hippocampus 64 days post-SCI. p. Representative images of hippocampal sagittal sections from SCI + bOECs, stained with anti-Iba1 and anti-CD86 antibodies. Arrows indicate Iba1+/CD86+ microglial cells. Scale bar = 100 µm. q. Representative images of hippocampal sagittal sections from SCI + bOECs, stained with anti-Iba1 and anti-CD206 antibodies. Arrows indicate Iba1+/CD206+ microglial cells. Scale bar = 100 µm. r. Quantification of microglial cell density (Iba1+) in the hippocampus and 64 days post-SCI. s. Quantification of microglial cells (Iba1+/CD86+) in the hippocampus 64 days post-SCI. t. Quantification of microglial cells (Iba1+/CD206+) in the hippocampus 64 days post-SCI. The green dotted line represents the values obtained from non-injured animals (d, e, f, g, h, i, j, m, n, r, s and t). Quantifications are expressed as mean ± SD (b, d, e, f, g, h, i, j, m, n, r, s and t). N = 4 (b), 5-6 (d-i), 6-8 (j), 6-9 (m, n, r, s and t) animals per group. Statistical evaluations were based on Mann-Whitney tests. * = P < 0.05; ** = P < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

<figure><img src="image_29.png" /></figure>

novel objects was measured using a camera positioned above the field (testing session) (Fig. 1c and 2c). Since mice have an inherent preference for exploring novel objects, a preference for the novel object indicates intact memory for the familiar object. The exploratory behavior of mice is evaluated by analyzing their interactions with objects and the areas containing objects, in comparison to areas without objects (Ishikawa et al., 2024). These experiments were conducted 32 and 64 days after SCI. Moreover, since this test can be influenced by the locomotor abilities of the animals, we also measured the total distance traveled during the test.

## 2.9. Tissue preparation and sectioning

Thirty minutes before euthanasia, mice received analgesic treatment to minimize pain (Buprenorphine 0.17 mg/kg). Following this, mice were deeply anesthetized with 5 % isoflurane and euthanized via intraperitoneal injection of Euthoxin (100 mg/kg). Transcardial perfusion with cold PBS was followed by ice-cold 4 % paraformaldehyde (PFA, Sigma Aldrich, 252549) in PBS. The dissected spinal cord and brain were then post-fixed in 4 % PFA overnight and cryoprotected in 30 % sucrose (Life Technologies, Carlsbad, CA) for at least 48 h. After embedding in Tissue-Tek OCT compound (Sakura, Tokyo, Japan), brains were cut sagittally into 30 µm thick sections. Sections were collected on slides and stored at -20 °C until further use.

## 2.10. Immunohistochemistry

Identification of different cells and structures in the brain was performed using primary antibodies diluted in a PBS-blocking solution containing 10 % normal donkey serum (Jackson ImmunoResearch, Cambridge, UK) and 0.3 % Triton-X100 (Sigma-Aldrich) to block non-specific binding. Antibody incubation was conducted overnight at room temperature in a humidified chamber. The following primary antibodies were used: mouse anti-glial fibrillary acidic protein (GFAP, Cy3-conjugated Sigma-Aldrich, C9205, RRID:AB_476889), rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1, Wako, 019-19741, Osaka, Japan, RRID), rat anti-B-lymphocyte activation antigen B7-2 (CD86, Invitrogen, MA1-10299, RRID:AB_11152324), goat anti-mMMR/CD206 (CD206, R&D systems, AF2535, RRID:AB_2063012), mouse anti-RNA binding Fox-1 homolog 3 (NeuN, Sigma, HPA030790, RRID) and goat anti-doublecortin (DCX, Santa Cruz, SC-8066, RRID).

After washing, antibody staining was revealed using species-specific fluorescence-conjugated secondary antibodies (Jackson

ImmunoResearch). Sections were counterstained with 4',6-diamidino-2-phenylindole (DAPI; 1 µg/mL; Sigma-Aldrich) and coverslipped with Vectashield mounting media (Vector Labs, Burlingame, UK).

## 2.11. Image acquisition analysis

Representative images for quantification of immunohistochemically stained areas in sagittal sections were captured using the Zeiss Apo-tome2 and Leica THUNDER Imager Tissue 3D microscope setups. For brain analysis, hippocampus images were obtained using a 20x objective lens. Image processing and assembly were performed using ImageJ software.

## 2.12. Quantification of immunohistochemically stained areas

The depth and number of brain sections analyzed were determined based on the depth and thickness of the granular cell layer in the OB. In the hippocampal sections, GFAP+, Iba1+, CD86+, CD206+, DCX+, and NeuN + cells were counted using standardized rectangular regions for each hippocampal area. For the dentate gyrus (DG) and cornu Ammonis (CA)1, cells were counted within rectangles of 500 µm × 250 µm. For the CA2 and CA3 regions, cells were counted within rectangles of 250 µm × 250 µm.

## 2.13. Statistical analysis

All data are presented as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism software, version 8.0.1.244 for Windows (GraphPad Software). The comparison of means was conducted using the two-way Mann-Whitney test. Detailed statistical analyses for each assay are provided in the figure legends. A p-value of < 0.05 was considered statistically significant.

# 3. Results

## 3.1. Transplantation of bOECs into the spinal cord improves cognitive behavior in mice after SCI

As previously described, the emergence of the cerebral inflammatory response after SCI is linked to cognitive deficits causing memory impairment in mice (Li et al., 2020). In a previous study, we demonstrated that spinal cord transplantation of bOECs reduced the inflammatory response in the spinal cord parenchyma (Delarue et al., 2021).
a.

A - No SCI group

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B - SCI group

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C - SCI + MSCs group

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Three cell passingag

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b.

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<table><thead><tr><th></th><th>Mean</th><th>SD</th></tr></thead><tbody><tr><td>Mesenchymal Stem Cells - CD105</td><td>87,8%</td><td>3,1%</td></tr><tr><td>Haematopoietic Cells - CD34</td><td>8,9%</td><td>2,4%</td></tr><tr><td>Endothelial Cells - CD31</td><td>3,1%</td><td>1,3%</td></tr></tbody></table>

c.

Training Session

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Testing Session

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d.

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e.

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f.

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l.

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n.

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o.

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(caption on next page)
**Fig. 2.** Transplantation of MSCs also corrects the cognitive deficit induced by SCI by acting on neurogenesis/neuronal survival and regulation of inflammation into the hippocampus. a. Schematic representation of the three experimental groups: A – No SCI group, B – SCI group, and C – SCI + MSCs group. b. Cytometric characterization of MSCs purified culture. Anti-CD105, anti-CD34 and anti-CD31 antibodies were used to identify MSCs, hematopoietic cells and endothelial cells, respectively. c-i. NOR experiments were conducted 64 days after SCI. c. Schematic representation of the NOR experiment. Like to bOECs transplantation experiment after a training session with two identical objects, one object was replaced by a new object different from the first. During the testing session, the frequency and duration of head orientation towards the new object were measured. d. Frequency of head orientation towards the familiar object at 64 days post-SCI. e. Frequency of head orientation towards the new object at 64 days post-SCI. f. Cumulative frequency of head orientation towards both objects at 64 days post-SCI. g. Duration of head orientation towards the familiar object at 64 days post-SCI. h. Duration of head orientation towards the new object at 64 days post-SCI. i. Cumulative duration of head orientation towards both objects at 64 days post-SCI. j-r. Immunohistology experiments. j and k. Quantification of mature neuron (NeuN+) density in the DG, CA3, CA2, and CA1 64 days post-SCI. j. Representative images of hippocampal sagittal sections from the SCI (j1) and SCI + MSCs (j2) groups, stained with anti-NeuN antibody. j1 and j2: Scale bar = 100 µm; DG, CA1, CA2, and CA3: Scale bar = 50 µm. k. Quantification of mature neuron (NeuN+) density in the DG, CA3, CA2, and CA1 64 days post-SCI. l and m. Analysis of astrocytes in the hippocampus 64 days post-SCI. l. Quantification of astrocytes density (GFAP+) in the hippocampus and 64 days post-SCI. m. Representative images of hippocampal sagittal sections from SCI + MSCs, stained with anti-GFAP antibodies. m1 and m2: Scale bar = 100 µm; m1' and m2': Scale bar = 50 µm. n-r. Analysis of microglial cell reactivity in the hippocampus 64 days post-SCI. n. Representative images of hippocampal sagittal sections from SCI + MSCs groups, stained with anti-Iba1 and anti-CD86 antibodies. Arrows indicate Iba1+/CD86 + microglial cells. Scale bar = 100 µm. o. Representative images of hippocampal sagittal sections from SCI + MSCs groups, stained with anti-Iba1 and anti-CD206 antibodies. Arrows indicate Iba1+/CD206+ microglial cells. Scale bar = 100 µm. p. Quantification of microglial cell density (Iba1+) in the hippocampus at 64 days post-SCI. q. Quantification of microglial cells (Iba1+/CD86+) in the hippocampus at 64 days post-SCI. r. Quantification of microglial cells (Iba1+/CD206+) in the hippocampus at 64 days post-SCI. The green dotted line represents the values obtained from non-injured animals (d, e, f, g, h, i, k, l, p, q and r). Quantifications are expressed as mean ± SD (b, d, e, f, g, h, i, k, l, p, q and r). N = 5 (b), 6 (d-i) and 8-9 (k, l, p, q and r) animals per group. Statistical evaluations were based on Mann-Whitney tests. * = P < 0.05; ** = P < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Based on these results, we have investigated the effects of bOECs transplantation in spinal cord after SCI on memory and exploratory behaviors, hippocampal neurogenesis, gliosis and inflammation. First, we performed flow cytometry cellular characterization of the primary culture of bOECs obtained from the OB of mice. Using PDGFRβ and P75 antibodies to identify stromal cells and OECs respectively, we found that our primary cultures consist of 63.8 % ± 6,9% stromal cells and 31.4 % ± 6,6 OECs. OECs are divided into two subpopulations: p75 high and p75 low OECs, representing 28.5 % ± 5,5% and 5.9 % ± 1,1%, respectively (Fig. 1b). These findings are consistent with previous studies on murine models, which have shown that primary cultures of bOECs consist of approximately 60 % to 70 % stromal cells and 30 % to 40 % OECs (Gómez et al., 2018).

Then, SCI and bOECs transplantation were performed at the same time, and longitudinal NOR experiments were conducted (Supplementary Fig. 1a-e and k-m and Fig. 1c-i).

First, we used this cognitive test, based on the ability to recognize a novel object in the environment, to assess the memory capacity of mice 32 and 64 days after SCI. As previously described, NOR results indicate that SCI induces a significant decline in memory capacity, as evidenced by the reduced frequency and duration of head orientation towards the novel object in the SCI group compared to the non-injured control group (dashed lines) at both 32- and 64-days post-SCI (data not shown and Fig. 1e and h, respectively). Thirty-two days after the lesion, analysis of the NOR test revealed no significant differences between the SCI and bOECs groups (data not shown). Sixty-four days after SCI, our results show that bOECs transplantation significantly increases the frequency and duration of head orientation towards the familiar and novel objects (Fig. 1d, g, e and h respectively). To determine whether this could reflect an increase in exploratory behavior, we measured the frequency and duration of head orientation towards the objects in both study groups at 64 days post-SCI (Fig. 1f and i). These parameters were significantly higher in the bOECs group compared to the SCI group (Fig. 1f and i). To further investigate whether this increase was indeed related to exploratory behavior, we measured the number of entries (frequency of entry) into the zones containing the objects in both groups. We found that mice in the bOECs group entered the object-containing zones 2.5 times more often than control animals (p = 0.07793), a behavior that was not observed in zones without objects (Supplementary Fig. 1m and l). Since this test can be influenced by the locomotor abilities of the animals, we compared the total distance traveled by both groups during the test. Sixty-four days post-lesion, there was no difference in this parameter between the SCI and bOECs groups (Supplementary Fig. 1k). All these parameters have been also analyzed 32-days after SCI and bOECs transplantation and do not show any difference between groups

(Supplementary Fig. 1a-e).

To investigate the mechanisms underlying cognitive improvement in transplanted mice, we collected brains from mice that had performed the NOR test at 64 days post-SCI. Additionally, to study the effects of bOECs transplantation at earlier stages, we transplanted bOECs into other groups of mice after SCI and collected their brains either 14- or 32-days post-SCI.

Firstly, we investigated hippocampal neurogenesis, as adult hippocampal neurogenesis plays a crucial role in maintaining learning and memory mechanisms in mice (Berdugo-Vega et al., 2020). Using immunohistochemistry, we measured the number of immature (DCX+) and mature (NeuN+) neurons in four hippocampal regions: DG, CA1, CA2, and CA3. These analyses revealed that bOECs transplantation increases the number of DCX+ cells in the hippocampus 14 days after SCI (Fig. 1j and k). Furthermore, bOECs transplantation increases the number of NeuN+ cells specifically in the DG at 64 days post-SCI (Fig. 1l and m), with no effect observed at 32 days post-SCI (Supplementary Fig. 1f). These findings suggest a potential increase in neurogenesis during the first two weeks following the injury, which may lead to the generation of mature neurons in the transplanted mice.

In physiological conditions, adult neurogenesis in the hippocampus is modulated by glial cells, especially astrocytes and microglia (Cope and Gould, 2019). However, in an inflammatory state, reactive gliosis, negatively impacts neurogenesis. To investigate this, we measured astrocyte density in the hippocampus of SCI and bOEC-transplanted mice at 32- and 64-days post-SCI (Supplementary Fig. 1g and Fig. 1n and o respectively). At 32 days post-SCI, bOEC transplantation significantly reduced astrocyte density in CA1 and CA2, with a downward trend observed in DG (p = 0.0617) and CA3 (p = 0.0932) (Supplementary Fig. 1g). More interestingly, this analysis shows that 64 days after SCI, GFAP density tends to be increased in bOECs group in DG and CA2 (Fig. 1n and o).

The inflammatory response negatively impacts the astrogliosis and cognitive function of mice (Prieto et al., 2019). Therefore, we explored the effect of bOECs transplantation on microglial activation in the hippocampus. To do this, we used Iba1 and CD86 antibodies to quantify microglial cell reactivity through immunohistochemistry at 32 and 64 days post-SCI. The number of Iba1 + cells was reduced in the CA1 region at 32 days post-SCI (Supplementary Fig. 1h) and in the CA3 region at 64 days post-SCI (Fig. 1p and r) in bOEC-transplanted mice compared to non-transplanted controls. Additionally, bOEC transplantation reduced microglial activation at 64 days post-SCI by decreasing the number of Iba1+/CD86 + activated microglial cells in the CA2 and CA3 regions (Fig. 1p and s), whereas no effect was observed at 32 days post-SCI (Supplementary Fig. 1i).
We also quantified microglial cells and macrophages that can adopt an anti-inflammatory phenotype, typically associated with the regulation of inflammation, by measuring the number of Iba1+/CD206 + cells. No significant differences were observed at 32 or 64 days between the SCI and bOEC-transplanted groups (Supplementary Fig. 1j and Fig. 1q and t respectively). However, a trend toward an increase (p = 0.0609) in Iba1+/CD206 + cell numbers was noted in the CA2 region of bOEC-transplanted animals at 32 days post-lesion (Supplementary Fig. 1j). Altogether, these results demonstrate that the transplantation of bOECs modulates inflammation primarily by reducing the number of Iba1+/CD86 + cells and has little effect on the number of Iba1+/CD206 + cells.

In summary, our results demonstrate that bOEC transplantation can restore exploratory behavior, potentially by enhancing hippocampal neurogenesis in the DG through the reduction of microglial activation and astrocytic gliosis in the CA regions.

## 3.2. Improvement of cognitive functions after SCI is also facilitated by non-neural cells transplantation

To confirm whether the improvement in memory and cognitive capacities is enabled by the transplantation of other cells, particularly those of non-neural origin, we conducted the same experiments and analyses on mice that received MSCs derived from adipose tissue following SCI. To do this, after cell culture characterization we performed the NOR experiment, quantified the number of neurons, and assessed hippocampal microglial and astroglial reactivity 64 days after SCI.

MSCs were obtained from inguinal adipose tissue. After three passages, cells were characterized by flow cytometry using CD105, CD31, and CD34 antibodies to identify MSCs, endothelial cells, and hematopoietic cells, respectively. We identified 87.8 % MSCs, 3.1 % endothelial cells, and 8.9 % hematopoietic cells (Fig. 2b), which is consistent with previous studies (Luna et al., 2014).

We then transplanted MSCs into mice following SCI and conducted the NOR experiment. Interestingly, similar to the bOECs transplantation experiments, and based on the same analyses—frequency and duration of head orientation towards the familiar object, novel object, and both objects—MSC transplantation at 64 days post-SCI did not compensate for the memory deficit but instead increased the exploratory behavior induced by SCI (Fig. 2c-i). Similar to the animals transplanted with OECs (Fig. 1), the MSC-transplanted animals entered the object-containing zones twice as often as the control animals (p = 0.054), with no difference observed between the groups in terms of entries into the object-free zones or total distance traveled during the test (Supplementary Fig. 1p, o and n respectively).

Although we did not quantify the number of immature DCX + neurons 14 days after SCI, it is possible that the improvement in cognitive performance is also linked to the induction of hippocampal neurogenesis in mice transplanted with MSCs. Indeed, the number of mature NeuN + neurons is increased in the DG 64 days post-SCI and transplantation. Unlike mice transplanted with bOECs, we also observed an increase in the number of mature neurons in CA2 and CA3 (Fig. 2j and k).

Astrocyte density has been also compared between SCI and MSCs groups 64 days after SCI. This analysis shows that there is no difference between groups (Fig. 2l and m).

Regarding the effect of MSC transplantation on hippocampal inflammation and gliosis, we observed a reduction in the number of microglial cells in the CA3 region, identified by Iba1 (Fig. 2n and p). Additionally, the number of Iba1+/CD86 + cells in the CA2 and CA3 regions was decreased 64 days post-SCI in MSC-transplanted mice compared to non-transplanted controls (Fig. 2n and q), indicating a reduction in hippocampal microglial reactivity. This analysis was further complemented by quantifying the number of Iba1+/CD206 + cells 64 days post-SCI in both groups. Similar to bOEC transplantation, MSC transplantation did not affect the CD206 + microglial cell population 64 days after SCI (Fig. 2o and r).

These results highlight the ability of MSCs transplantation into the spinal cord after SCI, similar to bOECs transplantation, to improve cognitive deficits induced by SCI. These effects appear to be associated with an increase in hippocampal neuronal density as well as a reduction in microglial reactivity.

# 4. Discussion

## 4.1. Therapeutic effects of bOECs transplantation after SCI on brain reactivity and disturbances of the cognitive functions

The purpose of our study was to investigate the effect of cell transplantation, bOECs and MSCs, used as a treatment after SCI on brain reactivity and cognitive functions. As described above, SCI induces microglia brain activation particularly in hippocampus (Li et al., 2020). Studies of anxiety, depression and cognitive performances of patients suffering SCI, show that it induces brain perturbations lasting for months after the injury. The importance of these perturbations is correlated with the severity of SCI, conditioning the cognitive deficits after the traumatism. Patients with high cognitive deficits, induced by SCI, have the worst cognitive recovery (Craig et al., 2017; Maldonado-Bouchard et al., 2016). These perturbations are related to microglia activation, causing a reduction of neuronal density which is linked to a decrease of neurogenesis and an increase of neuronal death. In rodents, specific test as NOR, allow to investigate brain reactivity and cognitive performances (Li et al., 2020). Based on these different studies, we carried out cognitive test after SCI with bOECs transplantation, MSCs transplantation or without treatment. Our findings show for the first time that cellular therapy applied at spinal level modulates the brain reactivity induced by SCI. Indeed, bOECs and MSCs transplantation enhances cognitive performance, in particular exploratory behavior, 64 days after SCI, objectified by NOR test (Fig. 1c-i and 2c-i). We also compared the results obtained in the NOR experiments with the two cell therapies to determine whether a difference could be observed, particularly whether one cell therapy might induce better cognitive recovery in the animals. These comparisons did not reveal any clear differences between our two transplanted groups, bOECs or MSCs (Supplementary Fig. 1q-v).

To elucidate how bOECs transplantation can enhance cognitive performance, we have performed histological analyses of hippocampus to quantify neurogenesis at early stage. Neurogenesis in DG is perturbed by SCI and is enhanced by bOECs transplantation 14 days after SCI (Fig. 1j). Based on the time required for a neuron to mature, we hypothesize that these new immature DCX + neurons contribute to increase the number of mature NeuN + neurons observed 64 days after SCI, as we did not observe any difference in neuronal density in the DG 32 days after SCI (Fig. 1m and Supplementary Fig. 1f).

This hypothesis is further supported by the fact that neurogenesis allows the generation of new neurons exclusively in the DG (Llorens-Martín et al., 2015; Zhao et al., 2007).

To confirm the beneficial effects of bOECs transplantation on the brains of mice who underwent SCI, we analyzed the hippocampus of mice transplanted with MSCs 64 days after SCI. Interestingly, we also observed an increase in the density of mature neurons NeuN + in the DG, as well as in the CA2 and CA3 regions (Fig. 2k). This could possibly be explained by an increase in neuronal survival in the MSCs-transplanted mice.

To investigate other mechanisms which can modulate cognitive functions, we have assessed the hippocampal inflammatory and astrocytic responses.

First, we measured the astrocytic density (GFAP + cell density) 32 and 64 days after SCI (Supplementary Fig. 1g, Fig. 1n and Fig. 2l). At 32 days post-SCI, it appears that the lesion induces a significant increase in astrocyte density in the hippocampus in comparison to non-injured mice, which can be interpreted as astroglial reactivity; this reactivity was reduced in animals transplanted with bOECs (dashed lines for non-injured mice, Supplementary Fig. 1g). At the opposite, 64 days after SCI,
GFAP + cell density analysis does not show significant difference between transplanted (bOECs and MSCs) and SCI-control groups (Fig. 1n and Fig. 2l). However, we can observe a trend toward an increase in GFAP + cell density in bOECs animals at the DG and CA2 regions (Fig. 1n). This trend toward increased astrocytic density in bOECs animals at 64 days post-SCI may be correlated with the increased neuronal density/neurogenesis observed in this group (Fig. 1n). Indeed, it has been demonstrated that astrocytes modulate hippocampal neurogenesis; in particular, these cells contribute to the proliferation of stem cells, as well as to the survival of newly formed neurons and their integration into a functional network (Cope and Gould, 2019).

Microglial cells participate to hippocampal homeostasis by modulating neurogenesis, synaptogenesis and neuronal death (Li et al., 2020). That is why we then investigated microglial reactivity, as evidenced by the assessment of Iba1 + cell density and the counting of Iba1+/CD86 + and Iba1+/CD206 + cells in the hippocampus. Our results, show that bOECs transplantation reduces microglial activation in CA1 area at the beginning of the chronic stage (Supplementary Fig. 1h). CA1 area is the starting point for neuronal projections leaving the hippocampus, it is important for memory transmission in other brain areas. Thus, the disruption of CA1 area brings memory deficits, particularly for non-spatial learning as object recognition, studied with NOR test (Basu and Siegelbaum, 2015). However, we did not find significative differences in NOR test results at 32 days post-SCI (Supplementary Fig. 1a-e). On the other hand, at chronic stage, bOECs and MSCs transplantation has no effect on microglial reactivity in CA1 but reduces it in CA3 (Fig. 1r and s and Fig. 2p and q). CA3 area is involved in episodic memory, encoding spatial representation and the coordination of hippocampal areas (Cherubini and Miles, 2015). Moreover, CA3-CA1 synaptic connections control long-term potentiation (LTP) generation in CA1 neurons. LTP generation in CA1 is correlated with learning and memory (Whitlock et al., 2006). Our analyses also show that the modulation of inflammation following the transplantation of bOECs and MSCs primarily occurs through the reduction in the number of Iba1+/CD86 + cells rather than an increase in the number of Iba1+/CD206 + cells (Fig. 1t and Fig. 2r). Indeed, the analysis of Iba1+/CD206 + cell numbers revealed no significant differences between our study groups 64 days post-SCI (Fig. 1t and Fig. 2r).

Altogether, our results show that bOECs and MSCs transplantation in spinal cord after injury induces therapeutic effects on cognitive deficits caused by SCI. This effect is potentially associated with improvement of neurogenesis at early stage based on the increase of the number of DCX + neurons. Moreover, bOECs and MSCs transplantation enhances the number of mature neurons (NeuN + ) at chronic stage (Fig. 1m and 2 k). These results regarding mature neurons can be interpreted as an improvement of neuronal integration of the newborn neurons (DCX + ) observed 14 days after SCI. Finally, our results show also that bOECs and MSCs transplantation decreases microglial cells reactivity in CA1 and CA3, favorizing the synaptic plasticity and learning.

This study does not allow for a precise description of the mechanisms involved, but several hypotheses can be proposed. Notably, in our study, it appears that the modulation of inflammation and neuronal survival/neurogenesis in the hippocampus are not directly linked within the same hippocampal regions (DG, CA1, CA2, and CA3), and we do not have specific mechanisms to explain this observation. However, we can hypothesize that although hippocampal neurogenesis is restricted to the DG, the hippocampus as a whole is composed of four distinct regions which are interconnected through direct and indirect projections, forming complex networks. These networks play a crucial role in cognitive functions such as memory and spatial navigation. Neurogenesis in the DG influences the activity of the CA regions of the hippocampus, and conversely, interactions between the CA regions and the DG also modulate neurogenesis in the latter. Inflammation within the hippocampus can disrupt these circuits by directly altering the function of neurons and glial cells in the inflamed region. Additionally, it can affect the projections between different regions of the hippocampus,

thereby influencing neurogenesis and associated functions. The cytokines and other diffusible factors secreted by inflammatory cells can act at distance, influencing cells located in other parts of the hippocampus. Therefore, we can hypothesize that inflammation or its regulation in a specific region, such as the CA3, could indirectly affect neurogenesis in the DG or the survival of neurons in other regions like CA1 or CA2.

As described above, one plausible explanation is the involvement of inflammatory cytokines propagation not only into the different regions of the hippocampus but also from the spinal cord to the brain. Indeed, cytokines, chemokines, and reactive oxygen species (ROS) are present in the cerebrospinal fluid (CSF) and blood after SCI, and they can increase the permeability of the blood-brain barrier (BBB), facilitating the infiltration of activated immune cells into the brain (Anthony et al., 2014; Capirossi et al., 2020; McCandless et al., 2008). Endothelial cells, which compose the BBB, express transporters for cytokines and chemokines (Banks et al., 1994; Osburg et al., 2002). Additionally, lymphocytes can transiently enter the brain through various routes, including the non-windowed capillaries of the choroid plexus. As lymphocytes constitute 50 % of immune cells in the CSF, they can be activated by cytokines in the CSF and, in turn, activate cerebral microglial cells (Ousman and Kubes, 2012). As we and others have demonstrated that bOECs transplantation after SCI reduces inflammation into the spinal cord (Delarue et al., 2021; Khankan et al., 2016; Zhang et al., 2021), we hypothesize that it decreases systemic inflammation, limiting the disruption of BBB and brain reactivity. This hypothesis is indirectly confirmed by the decrease of hippocampal microglial reactivity observed in bOECs transplanted animals. Although most of the transplanted cells die within the first three weeks post-transplantation, it is possible that during this period, bOECs modulate levels of cytokines and growth factors in the brain, thereby reducing disruption to neurogenesis. This modulation is likely due to both a direct effect, through the release of anti-inflammatory cytokines and growth factors, and an indirect effect by stimulating spinal cord regeneration, which in turn reduces the release of pro-inflammatory cytokines and immune cell activation.

## 4.2. Limitations of the study and conclusion

To our knowledge, this is the first study to demonstrate that cells transplanted into the spinal cord can modulate the inflammatory response in the brain, thereby reversing the deleterious effects of injury on brain function following SCI. Furthermore, our study highlights this effect in two distinct transplantation paradigms, showing that these benefits occur both after the transplantation of differentiated cells, such as bOECs, and after the transplantation of stem cells, such as MSCs.

However, in our study, while we observed an immunomodulatory effect of these two cell types on microglial reactivity, neuron density and astroglial reactivity in the brain, we were unable to elucidate the precise mechanisms underlying these results. Specifically, it remains unclear whether the transplanted cells exert their effects via the bloodstream or the CSF, or which cytokines are modulated by these transplants. Therefore, further experiments are needed to precisely identify the mechanisms involved.

Nevertheless, our findings underscore the complexity of the processes that occur following SCI and cell transplantation. They also open the door to a new and yet unexplored field of research into the interactions between the brain and spinal cord under both physiological and pathological conditions.

### Funding

This research was supported by ADIR association, IRME association, fonds de dotation Neuroglia and Fondation de l'Avenir.

### CRediT authorship contribution statement

**Quentin Delarue:** Writing – original draft, Validation, Methodology, Investigation, Data curation, Conceptualization. **Amandine Robac:** Methodology, Investigation, Data curation. **Fannie Semprez:**
**Methodology, Validation. Célia Duclos:** Methodology, Investigation. **Baptiste Pileyre:** Methodology, Investigation. **Pauline Neve:** Writing – original draft, Investigation. **Clémence Raimond:** Writing – original draft, Investigation. **Gaëtan Rou:** Validation, Methodology, Investiga- tion. **Inès Ziane:** Methodology. **Nicolas Guérout:** Writing – original draft, Supervision, Project administration, Funding acquisition, Formal analysis.

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.

Acknowledgements

Behavioural studies and processing were conducted with the support of equipment from the Behavioural Analysis Platform SCAC (University of Rouen Normandy, France).

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbi.2025.01.014.

Data availability

Data will be made available on request.

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