Immediate administration of hTERT-MSCs-IDO1-EVs reduces hypoalbuminemia after spinal cord injury

Abstract

Spinal cord injury (SCI) presents challenging and unpredictable neurological recovery. During inflammatory conditions, the amount of serum albumin and nutrition consumption decreases. Currently, it is proposed to measure serum albumin and glucose content in human or animal subjects to predict the recovery rate and the efficiency of treatments following SCI. In this study, the effect of extra-cellular vesicles (EVs) from immortalized human adipose tissue-derived mesenchymal stem cells (hTERT-MSCs) equipped with the ectopic expression of the human indoleamine 2,3-dioxygenase-1 (IDO1) gene on serum albumin and glucose levels was investigated. After pre-clearing steps of 72-hr conditioned media, small EVs (sEVs) were isolated based on the ultra-filtration method. They were encapsulated with a chitosan-based hydrogel. Five experimental groups (female rats, N = 30, ~ 230 g) were considered, including SCI, sham, hydrogel, control green fluorescent protein (GFP)-EVs and IDO1-EVs. The 60.00 µL of hydrogel or hydrogels containing 100 µg sEVs from GFP or IDO1-EVs were locally injected immediately after SCI (laminectomy of the T10 vertebra and clip compression). After 8 weeks, non-fasting serum glucose and albumin levels were measured. The results indicated that the level of serum albumin in the animals received IDO1-EVs (3.52 ± 0.04) was increased in comparison with the SCI group (3.00 ± 0.94). Also, these animals indicated higher glucose levels in their serum (250.17 ± 69.61) in comparison with SCI ones (214 ± 45.34). Although these changes were not statistically significant, they could be considered as evidence for the beneficial effects of IDO1-EVs administration in the context of SCI to reduce hypoalbuminemia and improve energy consumption. More detailed experiments are required to confirm these results.

Introduction

Complications in the secondary phase of spinal cord injury (SCI) include a series of inflammatory responses, glial scar formation, site-specific axonal degeneration¹ and organ dysfunctions, as well as motor, sensory, urinary and autonomic nerve disorders below the injury site.² It is relatively common for patients or animals with SCI to experience pressure sores, infections, and nutritional changes such as malnutrition, over-nutrition, protein-energy malnutrition and energy failure. These factors make individuals more susceptible to hypoalbuminemia (HA) and reduced metabolic rate.³ Following injury, spinal cord-specific cellular components such as enzymes and proteins are released into the bloodstream and cause hematological and biochemical changes.⁴ Therefore, the measurement of hematological and biochemical markers, such as albumin, can potentially be used to predict functional recovery and effectiveness of the therapeutic interventions.^5-7

The most abundant protein in blood plasma is albumin, which primarily acts as a blood transporter and helps maintain normal plasma oncotic pressure.⁸ Albumin is proposed as a suitable marker for assessing malnutrition and certain inflammatory or chronic diseases.⁹

There are several factors that can cause HA (abnormal and low concentration of blood albumin). These factors include malnutrition, reduced synthesis due to liver dysfunction, and increased catabolism following infection. Also, inflammatory status may lead to the leakage of albumin from the vascular system to extra-vascular spaces, which in turn increases trans-capillary escape rate of albumin. In addition, the leakage of plasma from the ulcer site in the form of sweat causes albumin loss.¹⁰ Studies have shown that HA plays an important role in predicting morbidity and mortality in major diseases and various clinical situations.¹¹ It has also been reported that more severe SCI and poor neurological recovery can be associated with lower concentrations of serum albumin soon after the injury.⁵ In addition, HA may reflect hyper-inflammation, reduced nutrition, and a decline in vital functions. It may also be due to the hyper-metabolic state associated with SCI, which may be exacerbated after surgical procedures.¹² However, the exact relationship between HA and poor outcomes has not yet been fully elucidated.

Today, the treatment of SCI remains one of the challenges for clinicians, and the effectiveness of common drugs is still controversial.¹³ Cell transplantation is considered as a novel therapeutic approach to manage diverse complications associated with SCI.¹⁴ Mesenchymal stem cells (MSCs) are multi-potent cells that can be isolated from several sources, including bone marrow, adipose tissue, umbilical cord, etc.¹⁵ Among these sources, adipose tissue is more accessible during liposuction¹⁶ and its collection is accompanied by minimal pain and invasiveness. These cells have anti-inflammatory effects and secrete different cytokines and growth factors which are important mediators of the immune system’s function. Indoleamine 2,3-dioxygenase-1 (IDO1), nitric oxide (NO), prostaglandin-E2, transforming growth factor beta 1 (TGF-β1),¹⁶ hepatocyte growth factor and interleukin-10 have been identified as the most efficient ones.¹⁷ The IDO1 is the rate-limiting enzyme in the tryptophan catabolic pathway and regarded as one of the key modulators of acquired immune tolerance.¹⁸ The immunomodulatory role of IDO1 has been reported in several publications.¹⁹

Although, the application of MSCs is associated with some limitations, such as the risk of chromosomal and genomic instability, potential risk of malignancies and altered functional properties.²⁰ Additionally, MSCs cannot cross the blood-brain barrier to reach the lesion site.²¹ Over the past decades, these limitations have led cell-free therapies to replace cell-based therapies. Cell-free therapies can be obtained by applying active components of the stem cells’ supernatant, mainly their extra-cellular vesicles (EVs).²² The application of EVs as one of the main mediators of the paracrine effects of MSCs has gained special interest.²³ The EVs are biological nanoscale spherical lipid bilayer vesicles being secreted by most cell types. Exosomes are one of the main EV sub-types, with a diameter of 40.00 - 100 nm.²⁴ The EVs are classified based on different criteria, among which their size and biogenesis are more common. ²⁵ Here, in accordance with the International Society for Extra-cellular Vesicles guidelines, the term small EVs (sEVs) is used to define the sub-population of EVs we applied following the SCI induction.²⁶ Due to their nanoscale dimensions, these membranous vesicles can easily cross the blood-spinal cord barriers and therefore, exert their immuno-modulatory properties, promote axonal regeneration and reduce neuroinflammation.²⁷

Various administration routes including intravenous, intrathecal and local injections have been proposed to deliver EVs to the lesion site. Hydrogels have been suggested as ideal scaffolds for use in spinal cord contusions because they can replicate the mechanical properties of natural spinal cord tissue.²⁸ Moreover, their application may increase the durability of EVs at the site of injury, and will guarantee the slow and stable release of their contents.^{21, 27, 28}

In this study, we aimed to determine if administering MSCs-EVs enriched from the conditioned medium (CM) of human telomerase reverse transcriptase (hTERT)-MSCs-IDO1 could decrease HA and increase the energy source in the plasma of the animal model. This is important considering the proposed beneficial effects of MSCs-EVs administration following SCI and the prognostic value of serum albumin and glucose concentrations.

Materials and Methods

Preparation of modified hTERT-MSCs. In this study, hTERT-immortalized adipose tissue-derived MSCs were used,²⁹ in which the human IDO1 gene was over-expressed based on the lentiviral approach. Cells which were transduced with the backbone plasmid lacking the IDO1 gene as the insert, hTERT-MSCs-green fluorescent protein (GFP), were used as a control for the main experimental group (hTERT-MSCs-IDO1).³⁰ Dulbecco's Modified Eagle Medium (DMEM)/F12 Medium (Gibco, Paisley, Scotland) containing puromycin (1.00 µg mL^-1; BioBasic, Toronto, Canada) was applied as a basal medium following the transduction steps (humidified incubator, 5.00% CO₂, 37.00 ˚C; Memmert, Schwabach, Germany). Cells were seeded in T175 cell culture flasks, and upon reaching the appropriate confluency (90.00 - 95.00%), they were sub-cultured (trypsin/ethylenediaminetetraacetic acid 0.25%; Gibco, Darmstadt, Germany) or stored for further applications. Control GFP or IDO1 transduced cells were selected to be used as EV producing cells. The preparation of genetically engineered hTERT-MSCs was performed in accordance with the ethical guidelines of the National Institute for Medical Research Development. These guidelines were aligned with the World Medical Association Declaration of Helsinki (Ethics Code: IRNIMADREC1396040).

Ultra-filtration. The hTERT-MSCs-GFP and hTERT-MSCs-IDO1 were seeded in equal amounts in T175 cell culture flasks (2.00 × 10⁶ cells per flask; SPL, Pocheon-si, South Korea). 48 hr later, their medium was removed and the basal DMEM/F12 medium supplemented with Exo-depleted Fetal Bovine Serum (10.00%)³¹ was added to the culture vessels following two rounds of washing with phosphate-buffered saline (PBS; 1.00X). At two intervals of 72 hr, the CM was collected, pre-cleared with centrifugation (400 g for 6 min and 1,500 g for 30 min, 4.00 ˚C; Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany) and syringe filtration (0.45 and 0.22 μm filters), and pooled. The prepared CM was used for sEV isolation immediately; otherwise, it was kept at –80.00 ˚C freezer. Isolation of sEVs (30.00 - 200 nm) was performed based on the application of a home-made ultra-filtration device. The ultra-filtration device, filtration-based device for exosome isolation from biological samples, being applied in the current project was registered in the Intellectual Property Center of Iran under Declaration Number Of 140050140003008723 on 7 February 2022 (Patent No. 108718, International Classification C12M 1/12; C12M 1/00). The isolated EVs were directly applied for down-stream characterization steps or animal studies. Under aseptic conditions, 100 µg of IDO1 or GFP-EVs were dissolved in a hydroxyl ethyl cellulose solution (Fluka, Seelze, Germany) and then added to a mixture of β-glycerol phosphate (Sigma Aldrich, Darmstadt, Germany) and chitosan (Polysciences, Warrington, UK) while kept on ice.³² Hydrogel containing insulin syringes or syringes filled with sEV-containing hydrogels (60.00 µL per insulin syringe per rat) were prepared and stored under cold aseptic conditions to be administered at the site of rat SCI during surgery procedures.

Bicinchoninic acid (BCA) assay. To determine the protein content of sEV preparations, the BCA assay was used according to the kit’s instructions (Parstous, Mashhad, Iran). A serial dilution of bovine serum albumin was applied as a standard for protein concentrations. Optical densities were measured at 545 nm after a 60 min incubation at 60.00 ˚C using an ELISA Reader (Awareness Technology, Palm City, USA).

Dynamic light scattering (DLS). Particle size analysis (PSA) was performed based on DLS. Size distribution of samples was measured by a particle size analyzer (Vasco 3, Cordouan Technologies, Cité de la Photonique, France).

Zeta potential analysis. Zeta potential was measured to investigate the stability and physicochemical properties of the samples (Zeta Compact; CAD Instruments, Naucelle, France). To perform the measurement, the samples were diluted (1:80) in PBS and the measurement was conducted at pH of 7.40 and room temperature. Each sample was subjected to at least three independent measurements.

Induction of SCI. All ethical principles were considered during the animal studies and approved by the Ethics Committee of Ferdowsi University of Mashhad, Mashhad, Iran (Ethics Code: IR.UM.RES.1401.024). The Ethics Code is available on the website of the Iran National Committee for Ethics in Biomedical Research. In this study, 30 female Wistar rats (age: 12 weeks and weight: 200 - 250 g) were randomly divided into five groups including SCI, sham, hydrogel, hTERT-MSCs-GFP-EVs and hTERT-MSCs-IDO1-EVs. The rats were anesthetized using an intramuscular injection of ketamine (70.00 mg kg^-1; Bremer Pharma GmbH, Warburg, Germany) and xylazine (5.00 mg kg^-1; Alfasan, Woerden, The Netherlands). To establish the animal model, we followed a previously published protocol with minor changes. Briefly, after clipping the hair on the rats’ back and surgically preparing the area, a 2.00 cm midline incision was made on the spinous process of T9-T10 vertebrae using a scalpel blade. The soft tissue and spinous process of T10 vertebra were removed. Laminectomy of T10 vertebra was performed to fully access the spinal cord. The drake aneurysm clip (Codman, Sheffield, UK) was placed on the spinal cord for one min.³³ Injection of 60.00 µL hydrogel or 60.00 µL hydrogel containing 100 µg sEVs was performed over the injured site immediately following the induction of SCI. The sham group only received laminectomy without SCI and did not receive any drugs (Fig. 1). Then, the muscles and skin were sutured using 3-0 vicryl (Supa, Tehran, Iran). Post-operative care includes the administration of 3.00 mL Ringer's solution (Samen Co., Mashhad, Iran) sub-cutaneously, intramuscular injection of tramadol (Darou Pakhsh Pharmaceutical Co., Tehran, Iran) at a dose of 10.00 mg kg^-1 every 12 hr for three days and sub-cutaneous injection of diluted enrofloxacin (Razak, Tehran, Iran) at a dose of 10.00 mg kg^-1 every 12 hr for one week. 

Fig. 1.

Schematic representation of the study. Small extra-cellular vesicles (sEVs) derived from hTERT-MSCs-GFP and hTERT-MSCs-IDO1 were embedded in modified chitosan hydrogel and injected at the site of injury (60.00 µL hydrogel or 60.00 µL hydrogel containing 100 µg sEVs) immediately following spinal cord injury (SCI) induction. After 8 weeks, the rats were sacrificed and their serum samples were used for downstream biochemical analysis measuring serum albumin and non-fasting glucose levels (created by BioRender.com). GFP: Green fluorescent protein; hTERT: Human telomerase reverse transcriptase; IDO1: Indoleamine 2,3 dioxygenase; MSCs: Mesenchymal stem/stromal cells

Bladder emptying through massage was performed twice a day until the return of bladder reflex activity, and physiotherapy and foot massage were also performed daily until the movement of the joints returned. The rats were maintained for 8 weeks in suitable conditions with free access to food and water.

Serum albumin and glucose measurement. After 8 weeks, under deep general anesthesia, intracardiac blood samples of the rats were taken under aseptic conditions and placed in plain tubes for serum separation. All tubes were immediately placed on ice and transferred to the laboratory. The plain tubes were centrifuged at 3,000 g for 10 min followed by removal of the serum. The serum was stored at – 80.00 ˚C until biochemical analyses. Finally, non-fasting glucose and albumin levels were measured using commercial kits (Pars Azmoon, Tehran, Iran) and an autoanalyser device (Targa 3000; Biotecnica, Rome, Italy).

Statistical analyses. The results were reported as mean ± standard deviation using SPSS Software (version 26.0; IBM Corp., Armonk, USA). Normality was assessed with the Kolmogorov-Smirnov test. Serum glucose was analyzed with one-way ANOVA and Tukey’s multiple comparisons post hoc test. Serum albumin was analyzed using the Mann-Whitney U test. A significance level of p < 0.05 was considered significant.

Results

Isolation and characterization of hTERT-MSCs-IDO1-EVs. The hTERT-MSCs-IDO1 and control hTERT-MSCs-GFP cell lines, which were used as EV producer cells were previously established³⁰ and their characteristics were determined based on the minimal criteria recommended by the International Society for Cell and Gene Therapy.³⁴ Figure 2 shows microscopic images of hTERT-MSCs before and after the transduction steps. As mentioned in the materials and methods, to isolate sEVs, the ultra-filtration device was applied following the pre-clearing steps of pooled CM from each experimental group.

Fig. 2.

Optical and fluorescent images of hTERT-MSCs. The images depict A) hTERT-MSCs before transduction, B and C) after the transduction steps. After the lentiviral transduction and the initial selection (2.00 µg mL^-1 puromycin, 72 hr), green fluorescent cells were maintained under a lower concentration of antibiotic for an additional 15 days. The hTERT-MSCs-GFP received lentiviral particles without IDO1 as an insert (Olympus, Tokyo, Japan; bars = 200 µm). GFP: Green fluorescent protein; hTERT: Human telomerase reverse transcriptase; IDO1: Indoleamine 2,3 dioxygenase; MSCs: Mesenchymal stem/stromal cells

The number of cells at the time of CM harvest was determined to be 2.70 × 10⁵ based on the formula suggested by Börger et al.³⁵ The total protein content of sEVs was determined to be 6008.78 µg mL^-1 for GFP-EVs and 7528.72 µg mL^-1 for IDO1-EVs based on the BCA assay. These concentrations were applied as a quantification criterion to normalize the amount of sEVs administered to each rat during injections. The size distribution of the particles was determined to be between 30.00 - 200 nm with number distribution size of 47.28, Z-average/harmonic intensity averaged particular diameter of 179.98, and poly dispersity index of 0.3500 via PSA analysis (Fig. 3A). Zeta potential analysis reported a mean zeta potential of – 25.07 ± 8.06 mV, indicating an acceptable level of stability for IDO1-EVs (temperature: 24.15 ˚C and pH: 7.80; Fig. 3B).

Fig. 3.

Particle size and zeta potential analyses. A) Particle size analysis based on dynamic light scattering confirmed desirable size range for extra-cellular vesicles (EVs) isolated based on the ultra-filtration method (mean: 47.28). B) Mean of zeta potential of – 25.07 mV was recorded for indoleamine 2,3 dioxygenase-EVs

Serum concentration of albumin. Evaluating the concentration of albumin in the blood serum of rats 8 weeks following the surgeries indicated that the amount of serum albumin in the SCI group was lower than other groups. This decreasing trend in serum albumin levels was observed in the hydrogel, GFP-EVs and IDO1-EVs groups, respectively (Table 1). However, these differences were not statistically significant (p > 0.05).

Table 1.

Comparison of serum albumin and glucose concentrations between different groups at 8 weeks post-surgery

Experimental groups	Albumin (g dL -1 )	Glucose (mg dL -1 )
SCI	3.00 ± 0.94	214.00 ± 45.34
Sham	3.63 ± 0.21	246.50 ± 36.72
Hydrogel	3.28 ± 0.21	220.17 ± 55.69
MSCs-GFP-EVs	3.48 ± 0.19	232.00 ± 31.44
MSCs- IDO1 -EVs	3.52 ± 0.04	250.17 ± 69.61

EVs: Extra-cellular vesicles; GFP: Green fluorescent protein; IDO1: Indoleamine 2,3 dioxygenase; MSCs: Mesenchymal stem/stromal cells; SCI: Spinal cord injury.

No significant difference was observed between groups in terms of serum albumin and glucose levels (p > 0.05).

Serum concentration of glucose. As shown in the Table 1, the SCI group had the lowest blood glucose level. Following that, the hydrogel and GFP-EVs groups had the lowest blood glucose levels, respectively. On the other hand, the IDO1-EVs group had the highest blood glucose level, even higher than the sham group. However, no statistically significant difference was observed (p > 0.05). The hTERT-MSCs-IDO1-EVs decreased HA in comparison with SCI, hydrogel and GFP-EVs groups. Serum glucose level in IDO1-EVs group was higher than other experimental groups.

Discussion

Primary SCI causes axonal damage and tissue necrosis. Following SCI, a cascade of secondary events including metabolic, biochemical and cellular changes, such as electrolyte abnormalities, lipid peroxidation, free radicals formation, NO action, ischemia, edema and apoptosis in the microcirculation occurs.³⁶ Additionally, there is an increased secretion of pro-inflammatory cytokines after SCI, leading to increased vascular permeability, dilution of serum albumin, accelerated removal of albumin from the circulation, and HA development. It is believed that HA may indicate a hostile pro-inflammatory state,³⁷ and could be a strong predictor of poor outcomes for various central nervous system (CNS) diseases and injuries, including traumatic brain³⁸ and chronic SCIs.¹² In patients with neurological damage, persistent reduction in albumin levels leads to organ function destruction, impairs recovery from spinal cord edema, and exacerbates blood supply, nutrition and metabolism of the nervous system. These events perpetuate a debilitating cycle.³⁹ On the other hand, several studies have demonstrated the neuroprotective role of albumin in CNS injuries.⁴⁰

The relationship between albumin, inflammation and neurological recovery has also been described in a previous study.⁵ As one of the main therapeutic goals after SCI is to modulate the inflammatory environment to promote neuronal regeneration and manage extensive secondary cell death, we used genetically modified hTERT-MSCs to modulate inflammatory events in a rat model of SCI. These cells retain essential features of primary cultures of MSCs including spindle-like morphology, adherence to tissue culture plastic, differentiation and growth capacity over several passage numbers, serving as an immortalized model cell line of MSCs.²⁹ Moreover, due to their origin, it can be assumed that they will have prominent immuno-regulatory properties reported for primary cultures of MSCs derived from adipose tissue.⁴¹ Additionally, hTERT immortalization has previously been suggested as a practical method for establishing of cell lines with a remarkable ability to prepare clinical-grade EVs.⁴²

In a previous study, we investigated the immune regulatory characteristics of hTERT-MSCs and their EV counterparts against human peripheral blood mono-nuclear cells (hPBMCs) and Jurkat cells. The cells were primed with different compounds or genetically modified using the lentiviral approach to over-express human IDO1, prostaglandin-endoperoxide synthase and TGF-β1 genes individually or concurrently in the co-transduced group. Results indicated that hTERT-MSCs-IDO1-EVs showed the highest proliferation inhibitory effects against allogenic hPBMCs.³⁰ Different catabolites, closely related to various diseases, are involved in the formation of the immunosuppressive environment, and different mechanisms have been proposed to describe the IDO1-mediated immunoregulation. This immunoregulation may be due to the lack of local tryptophan in the micro-environment, activating the general control nonderepressible 2 (GCN2) kinase and mammalian target of rapamycin pathways, as well as T cell anergy.⁴³ Consumption of tryptophan by IDO1 removes this essential amino acid required for the production of various proteins in the environment. Tryptophan deficiency activates GCN2 kinase in T cells leading to cell arrest in the G2 phase of the cell division cycle. Inhibition of T helper type 1 (Th1) cells by IDO1 causes a selective increase of Th2 cells response.⁴⁴ Additionally, when CD4⁺ T cells combine with IDO1, they become regulatory T (Treg) cells, inhibiting the activation, differentiation and survival of killer T cells. The Treg cell response induced by IDO1 inhibits both types of Th1 and Th2 cells; thereby, moderating the immune system. Moreover, IDO1 metabolites are capable of inhibiting the proliferation and function of natural killer cells. All these factors create an environment that helps to adjust and regulate the immune system. ⁴⁵

Here, we hypothesize that hTERT-MSCs-IDO1-EVs may prevent the spread of secondary damage following SCI by modulating the immune system’s activity. This is based on their anti-inflammatory and immuno-modulatory properties, controlling the inflammatory environment and inhibiting the inflammatory factors expression.⁴⁶ As a proof of concept, we demonstrated that the immediate administration of hTERT-MSCs-IDO1-EVs in a xenogeneic rat model of SCI improves the functional recovery of rats in comparison with other experimental groups (data not shown). Additionally, hTERT-MSCs-IDO1-EVs are indicated to reduce HA and improve nutrition after SCI.

The beneficial effects of albumin in the CNS include the reduction of free radicals, platelet aggregation, leukocyte adhesion and histological damage to neurons, prevention of capillary stasis and NO regulation. Moreover, albumin has been shown to improve metabolic activity of neurons and micro-circulatory flow in animal studies.³⁹ Recent studies have also shown that prolonged HA is proportional to a lesser neurological disease,^5,7 inflammation, infection, chronic diseases,⁴⁷ patient malnutrition⁹ and poor health, as well as an increased risk of mechanical ventilation and mortality after acute SCI.³⁹ It has been reported that intra-peritoneal treatment with human serum albumin improves early clinical results, protects spinal cord ultra-structure, and decreases myeloperoxidase activity and lipid peroxidation levels after spinal cord contusion injury in rats in comparison with methylprednisolone.⁴⁰

Leister et al. have shown that routine blood chemistry data from post-acute phase, along with baseline injury severity can predict functional outcome after incomplete SCI.³⁷ Also, Vo et al. and Jin et al. have demonstrated that albumin concentration correlates with baseline characteristics and long-term neurological recovery after SCI. During rehabilitation, serum albumin concentration can also estimate the severity of injury and future neurological recovery.^{7, 39} In a study by Tong et al., the effect of monosialotetrahexo-sylganglioside on human serum albumin levels and motor performance improvement was investigated. It was found that higher serum albumin concentrations at 1, 2 and 4 week periods were associated with higher 52-week motor function scores. The researchers suggested that serum albumin could serve as a feasible biomarker for prognosis at the time of injury and stratification in clinical trials.⁵

The main source of energy for the CNS is glucose, which ensures the necessary energy for neural activity.⁴⁸ Disturbances in glucose metabolism lead to the occurrence and development of various diseases related to the nervous system.⁴⁹ It has been concluded that remarkable reduction of whole body glucose is mainly due to the reduction in muscle mass (atrophy), and that skeletal muscles have a remarkable capacity to maintain their glucose transport system intact even in the face of severe denervation and morphological changes.⁵⁰

In the present study, the glucose levels were measured in a non-fasting state to monitor its levels under normal conditions. Similar to serum albumin levels, the SCI group showed the lowest blood glucose level compared to the other groups. The hydrogel and GFP-EVs groups had higher serum glucose levels than SCI group; but, lower than the IDO1-EVs group. We hypothesize that the lack of improvement in the nervous system in the absence of any treatment schedule and the subsequent decrease in the movement capacity/score along with factors such as infection and stress may lead to a decrease in serum albumin and blood glucose levels. However, further investigations are required to attribute these results to the human cases.

Biochemical parameters can be assessed repeatedly in the first days or weeks after SCI without the need for invasive procedures. This evaluation is quick and inexpensive. The levels of these parameters are directly related to the severity of injury and the extent of neurological recovery. In our study, we utilized the measurement of serum albumin and glucose levels to assess the effectiveness of hTERT-MSCs-IDO1-EVs in a rat model of SCI. We propose that EVs derived from the CM of hTERT-MSCs-IDO1 can replicate the immuno-modulatory characteristics of their parent cells and their anti-inflammatory effects can be detected through simple blood tests.

Conflict of interest

The authors declare no conflict of interest.