**1. Introduction**

Lesions of the spinal cord due to traumatic injury, tumors, or vascular ischemia frequently cause paralysis and the loss of autonomic functions. No disease modifying therapy for this pathology is currently available. The severe consequences of spinal cord injury (SCI) are in a large part due to a secondary inflammatory reaction, which is borne by local microglia cells, macrophages, and lymphocytes that infiltrate the lesion area. This reaction, in conjunction with increasing vascular permeability, causes cell death of neurons and glia cells [1]. For this reason, anti-inflammatory treatment is a therapeutic strategy for SCI and has been used in the clinic [2]. Unfortunately, the approved treatment option, methylprednisolone, is often ineffective and causes severe side effects such as a higher incidence of sepsis, gastrointestinal hemorrhage, or pulmonary embolism [3]. Therefore, new therapies for SCI are needed, and one of the most promising lines of research with this aim consists of the application of stem cells [4–6]. We have recently tested the safety and therapeutic benefits of human bone marrow-derived stromal cells (bmSC), which are prepared solely by negative selection without expansion in vitro (Neuroplast BV, patent WO2015/059300A1).

**Citation:** Wu, S.; García-Rama, C.; Romero-Ramírez, L.; de Munter, J.P.J.M.; Wolters, E.C.; Kramer, B.W.; Mey, J. Tauroursodeoxycholic Acid Reduces Neuroinflammation but Does Not Support Long Term Functional Recovery of Rats with Spinal Cord Injury. *Biomedicines* **2022**, *10*, 1501. https://doi.org/10.3390/ biomedicines10071501

Academic Editor: Krisztina Nikovics

Received: 4 April 2022 Accepted: 23 June 2022 Published: 25 June 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

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In rat models of SCI, intrathecal infusion of these cells reduced chronic inflammation and neural degeneration and provided a benefit on the functional level [7,8]. However, as in most other studies [5], the beneficial effect was limited. In comparison with control treatments, the recovery of sensory-motor function improved by 1.5 points (BBB scale) [9], and even at nine weeks after SCI, most rats did not recover beyond BBB 9-11 [8]. One reason for the limited effect of bmSC is that implanted cells die or are actively eliminated in the acute phase. We reasoned that a combinatorial therapy of bmSC implantation with additional cytoprotective measures would be advantageous to support the integration of implanted bmSC.

A recent strategy of cytoprotection for neuropathologies is based on bile acids, which have long been used in traditional Chinese medicine [10,11]. These are amphipathic molecules synthesized from cholesterol. Their biological effects are mediated via the Takeda G protein-coupled receptor-5 (TGR5) [12,13] as well as nuclear receptors farnesol X receptor, pregnane receptor, and liver X receptors [14,15]. While bile acids play important roles in lipid metabolism [16], they also have anti-inflammatory and cytoprotective effects by suppressing NFκB signaling, which make them interesting candidates for the treatment of neuropathologies [17,18]. One particular bile acid, tauroursodeoxycholic acid (TUDCA), has been tested in animal models for Parkinson's disease [19], multiple sclerosis [20], and a clinical trial of amyotrophic lateral sclerosis [21]. In rodent models of SCI, TUDCA reduced cellular apoptosis [22–26]. With rats, improvements on the functional level were observed within the first 5 days after injury, i.e., in the subacute phase, but it is not clear whether these treatments have a lasting effect after SCI.

Our objectives in the present study were (1) to assess the effects of TUDCA on neuroinflammation and astrogliosis after SCI, (2) to clarify the long term therapeutic effects of TUDCA treatment on the recovery of sensory-motor function, and (3) to test whether a combinatorial therapy of TUDCA and bmSC transplantation provides additional benefit.

### **2. Materials and Methods**

### *2.1. Experimental Animals and Study Design*

Experimental protocols, surgical procedures, and post-operational care were reviewed by the ethics committee for animal care of the Hospital Nacional de Parapléjicos (163CEEA/ 2017) and approved by the Consejería de Agricultura y Ganadería de Castilla-la Mancha (ref. 210498, following EU directive 2010/63/EU). We used male Wistar rats (*Rattus norwegicus*), six to eight weeks of age, which had been bred in the animal facility of the hospital. Until the day of surgery, animals were kept in pairs and, subsequently, in individual cages. Standard housing conditions consisted of a 12 h light/dark cycle, 40–60% humidity, temperature of 22 ◦C with ad libitum access to food and water. A total of 75 animals entered the study (Figure 1a; sample size calculation for BBB scores at 6 weeks based on α = 0.05, β = 0.2, d=3, SD = 2, attrition 10%). In addition, the spinal cords of non-injured rats were processed for comparison of histological results. Since rats had the same sex, and a similar age and body weight, no randomization was performed to allocate them in the study. The order of treatment was mixed to ensure that all treatment groups were served throughout the entire period when surgery was performed. Rats were excluded when the SCI was considered invalid. Since the primary outcome of the study was sensory-motor function, the exclusion criteria were a BBB score above 2 at 24 h after SCI or a force/time plot of the impactor device that indicated that bone was hit.

### *2.2. Surgical Procedures and Postoperative Treatment*

Spinal cord contusion injury and injections of bmSC were performed as described previously [8]. In short, anesthesia consisted of 2.5% isoflurane/97.5% oxygen at 0.5 L/min for SCI. For the injections of bmSC, we used one i.p. injection of ketamine 50 mg/kg combined with xylacine 5 mg/kg. Fifteen minutes before surgery, rats received one s.c. injection of buprenorfine 0.05 mg/kg to reduce pain. Corneal dehydration was prevented with ophthalmic ointment (Lubrithal, Dechra, Barcelona, Spain). With ketamine anesthesia,

0.04 mg/kg of atropin was given. Following laminectomy at thoracic level T9, a spinal cord contusion of 2 N (200 Kdyn, zero dwell time) was inflicted with the Infinite Horizon (IH) spinal cord impactor. We checked the procedure visually (hematoma) and by monitoring the IH displacement/time and force/time plots. To normalize biochemical data at 4 days post operation (dpo), a control group was operated on using the laminectomy procedure without SCI. Immediately after surgery, all animals received 2 × 2.5 mL of isotonic saline s.c. to prevent dehydration and antibiotic treatment with marbofloxacin 5 mg/kg (10 mg/mL s.c. Marbocyl, Alcobendas, Spain). Surgery and behavioral assessments were performed between 09:00 and 14:00.

The transplantation of bmSC was done 2 h after SCI. After the anesthetized animals were positioned in a stereotactic frame, the atlanto-occipital membrane was exposed and penetrated with a pointed scalpel blade. A catheter was then inserted and the cell suspension was slowly infused with a syringe pump (100 μL/3 min) into the cisterna magna. While a rat was being prepared by one researcher, a second person removed one batch of bmSC from storage in liquid nitrogen, thawed and washed the cells with saline, and resuspended them in 110 μL of saline. From this, 10 μL was removed for cytometric counting of cell numbers and determination of cell viability. On average, one injection of 100 μL contained 2.6 million viable cells.

Postoperative care, including analgesic and antibiotic treatment, was done as previously described [8]. The bladders were checked and voided manually every 12 h until the rats were urinating spontaneously. The volume of retained and manually expelled urine per 12 h was recorded. Euthanasia at the end of the study was induced by an i.p. injection of 100 mg/kg of sodium pentobarbital (Dolethal, Madrid, Spain).

### *2.3. Experimental Groups*

Animals were assigned to seven experimental groups, six of which received the same SCI but differed in the treatment procedure (Figure 1a). One group (sham) had T9 laminectomy but underwent no contusion injury. Group *SCI-control* received two i.p. injections of saline, the first immediately after SCI (t0) and the second 24 h later (1 dpo). Group *SCI-T200* was treated with two i.p. injections of TUDCA 100 mg/kg body weight at t0 and 1 dpo. Group *SCI-T600* had two i.p. injections of TUDCA 300 mg/kg at t0 and 1 dpo. Group *SCI-T1500* had five injections of TUDCA 300 mg/kg at t0, 1 dpo, 2 dpo, 4 d po, and 6 dpo. Group *SCI-bmSC* received human bmSC implants at t0 + 2 h. Group *SCI-bmSC + T* had bmSC at t0 + 2 h and, in addition, two injections of TUDCA 100 mg/kg at t0 and 1 dpo. During the following six weeks, the investigators who performed the behavioral evaluation were blind with regard to the experimental condition of the individual animals. Nine rats from each of the laminectomy (sham), SCI-control, and SCI-T600 groups were sacrificed at 4 dpo for biochemical and histological analysis. Additional spinal cord sections from non-injured rats were used for comparing histological data.

### *2.4. Preparation of bmSC*

Human bmSC for SCI treatment were prepared by negative selection eliminating erythrocytes with Ficoll density gradient centrifugation and, subsequently, B-cells (CD20), T-cells (CD3), monocytes (CD14) and natural killer cells (CD56) using antibody-based cell sorting with magnetic beads under GMP conditions. Cells were not expanded by cultivation (Neuroplast BV, patent WO2015/059300A1, Geleen, The Netherlands). All procedures for the collection of human bone marrow were approved by the ethics committee of Maastricht University Medical Center (METC 13-2-032). The viability and cell type composition of each batch were analyzed with flow cytometry (CD34, CD271, CD90, CD105, CD73). For the present study, bmSC were prepared at the Neuroplast facility in Geleen, NL, cryoprotected with DMSO, frozen in liquid nitrogen, shipped on dry ice to Toledo, Spain, and then stored in liquid nitrogen until use. Cell viability (exclusion of 7-amino-actinomycin D, cytometry) was again determined after thawing, i.e., immediately before application in vivo.

**Figure 1.** Experimental plan. (**a**) Animals were pseudo-randomly assigned to the different treatment groups. Functional analysis was carried out during a recovery period of six weeks after SCI with the following treatment conditions: (1) two injections of saline at the time of surgery (t0) and 24 h later, (2) two injections of 100 mg/kg TUDCA at t0, 24 h; (3) two injections of 300 mg/kg TUDCA at t0, 24 h; (4) five injections of 300 mg/kg TUDCA at t0, 24 h, 2 dpo, 4 dpo and 6 dpo; (5) one injection of bmSC at t0 + 2 h; (6) combinatorial treatment with bmSC and two injections of 100 mg/kg TUDCA. In addition, three treatment groups (laminectomy only, SCI-control, and SCI with 2 × 300 mg/kg TUDCA) were evaluated at 4 dpo with biochemistry and IF. In histology at 6 W, spinal cord tissue was also compared to tissue from non-lesioned rats. Two animals had to be excluded from the study because open field evaluation at 1 dpo suggested an incomplete lesion (red arrows a: BBB = 7, b: BBB = 8). (**b**,**<sup>c</sup>**) Changes in body weight following SCI: Animals in all treatment groups suffered from weight loss during the first 4 dpo and subsequently recovered [means +/− SD; see text for statistical evaluation].

### *2.5. Bile Acid Treatment*

Tauroursodeoxycholic acid (Calbiochem CAS 14605-22-2, Millipore, Madrid, Spain) was dissolved at 150 mg/mL in 0.9% saline immediately before the intraperitoneal injection or stored for no longer than 24 h at 4 ◦C.

### *2.6. Evaluation of Locomotor Functions*

This was the primary outcome of the study. Recovery of limb movements was evaluated in the open field using the Basso/Beattie/Bresnahan (BBB) locomotor function scale [9]. This was done before surgery (baseline), at 1 dpo, 2 dpo, 3 dpo, 4 dpo, 7 dpo, and subsequently once per week until six weeks after SCI. At the beginning, we established a criterion of BBB ≤ 2 at 1 dpo for inclusion in the study because a higher score was considered to indicate incomplete SCI. Scoring was performed independently by two investigators who were blind with respect to the treatment of the individual animals. Following assessment, both investigators discussed their evaluation, and in cases where different scores were given, the average of both was recorded.

A second assessment was made using the Rotarod test (Ugo Basile SRL, Gemonio, Italy). In this task, rats are positioned on a slowly rotating rod, which obliges them to use their hind legs in order to keep their balance [27]. In six training sessions of 5 min each, at three, two, and one days before SCI, all rats learned this task at a constant speed of 5 rpm of the rotating rod. During tests, which were administered at 7 dpo and then once per week, the rotation speed was accelerated from 5 rpm to 15 rpm over a period of 5 min. The readout in this assay was the time that the rats were able to stay on the rotating rod before falling off (two repetitions, separated by a break of ≥15 min). At 4 dpo, we confirmed that none of the SCI rats to be included in the study showed weight supported steps. Considering the high variability of this assay, we applied an additional performance assessment using the percentage of animals in each group that were able to maintain their balance for more than 30 s at 6 W after SCI.

### *2.7. Von Frey Test of Mechanical Allodynia/Hyperalgesia*

Tactile allodynia/hyperalgesia were evaluated using a dynamic plantar aesthesiometer (von Frey test; Hugo Basile 37550, Gemonio, Italy). For each hind leg, a paw withdrawal threshold (PWT) was determined up to a maximum force of 50 g. This was done five times, with intervals of at least five minutes between tests. The lowest and highest values of these readings were excluded, and then the mean was calculated as PWT. This test was administered five weeks after SCI, when all animals were physically able to respond to the stimulation. On the basis of measurements before the lesion, we considered a PWT of below 20 g as an indication of neurogenic pain.

### *2.8. Quantification of Gene Expression*

Animals for biochemical evaluation were sacrificed at 4 dpo with an overdose of sodium pentobarbital. After opening the thoracic cavity, a blood sample of 1 mL was taken from the heart, mixed with 100 μL 0.5 M EDTA to prevent coagulation, spun down, and the supernatant frozen (samples not intended for this study). This was followed by transcardial perfusion with phosphate buffered saline (PBS; 200 mL/rat), preparation of the brains and spinal cords. Spinal cord samples consisted of a 2 cm segmen<sup>t</sup> with the lesion site in the center. Tissues were homogenized mechanically with Trizol (Invitrogen, 15596018, Madrid, Spain) and the RNA extracted according to manufacturer's instructions. To remove genomic DNA, purified RNA was digested with DNase I (ThermoScientific, EN0521, Madrid, Spain). An aliquot corresponding to 0.5 μg of purified RNA was used for first-strand cDNA synthesis using Superscript III reverse transcriptase and oligo (dT) primers in a final volume of 40 μL (Invitrogen Life Technologies, K1632). A real-time quantification of cDNA was performed using a SYBR Green PCR assay. Each 15 μL SYBR green reaction mixture consisted of 1 μL cDNA, 7.5 μL SYBR Green PCR-mix (2×), 0.75 μL forward and reverse primers (10 pM) and 4.75 μL distilled water. PCR was performed with 5 min at 95 ◦C, followed by 40 cycles of 15 s at 95 ◦C, 60 s at 60 ◦C and a separate dissociation step for the melting curve. The specificity of the PCR product was confirmed by ascertaining a single melting peak in the temperature dissociation plots. All samples were run in triplicates and the level of expression of each gene was compared with the expression of acidic ribosomal phosphoprotein P0 (36B4). Amplification, detection of specific gene

products and quantitative analysis were performed using an ABI 7500 sequence detection system (Applied Biosystems, Alcobendas, Spain). PCR efficiency was verified by dilution series (1, 1/3, 1/9, 1/27, 1/81, and 1/243) and relative mRNA levels were calculated using the comparative ΔCt method with normalization to 36B4. Gene identifiers, primer sequences, product sizes, and melting temperatures are listed in Table 1.

**Table 1.** Primer sequences used in quantitative RT-PCR.


See list for abbreviations, all gene sequences are from rat.

### *2.9. Tissue Preparation and Histological Staining*

At four days or six weeks after SCI, rats were sacrificed with an overdose of sodium pentobarbital followed by transcardial perfusion with PBS and 4% paraformaldehyde/PBS (PFA). The spinal cords were prepared, post-fixed for 1 h, then stored at 4 ◦C in PFA for 1–3 days. For histological processing, 2 cm long spinal cord segments that included the lesion site in the center were dissected, dehydrated, embedded in paraffin, and cut in 3 μm parasagittal sections using a Leica RM2265 microtome. Sections were mounted on polylysine-coated glass slides (Superfrost Plus, Fisher Scientific, Madrid, Spain) and stored at room temperature (RT). Apoptotic cell nuclei were stained with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) using the One-step fluorescence TUNEL apoptosis kit (Elabscience, E-CK-A325, Houston TX, USA) according to the manufacturer's protocol.
