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BY 4.0 license Open Access Published by De Gruyter Open Access July 15, 2021

A meta-analysis of exosome in the treatment of spinal cord injury

  • Hanxiao Yi EMAIL logo and Yang Wang
From the journal Open Medicine

Abstract

Context

There are no recommended therapeutic agents for acute spinal cord injury (SCI) due to the pathophysiological complexity of the injury.

Objective

The objective of this study is to investigate the efficacy of various exosomes and potential factors impacting the efficacy of exosomes.

Methods

We searched the PubMed, EMBASE, Web of Science, Medline, Scopus, and Cochrane Library databases to systematically collect articles comparing the locomotor function of SCI rodents undergoing exosome treatment and untreated SCI rodents. No language was preferred.

Results

Pooled analysis revealed that the locomotor function recovery of SCI rodents receiving exosomes was greatly improved (583 rats, 3.12, 95% CI: 2.56–3.67, p < 0.01; 116 mice, 2.46, 95% CI: 1.20–3.72, p < 0.01) compared to those of control rodents. The trial sequential analysis demonstrated the findings of the meta-analysis with the cumulative Z-curve crossing the upper monitoring boundary for the benefit and reaching the adjusted required information size. However, the origin of the exosome, SCI model, and administration method determined the therapeutic effect to some extent.

Conclusions

Despite the proven therapeutic effects of exosomes on SCI rodents, the results should be interpreted cautiously considering the diversity in vivo and in vitro in relation to future trials.

1 Introduction

Spinal cord injury (SCI), a life-threatening disorder, is closely associated with deficits in locomotor function and sensation [1] and has an annual prevalence of 10–83 cases per million [2], with 90% of these cases being traumatic SCI. Early decompression is usually recommended for patients with SCI; however, postsurgical drug treatment strategies are still lacking.

Secondary inflammation after SCI directly induces extension of the injury, which is the result of ischemia, inflammation, secretion of excitotoxic substances, and worsening deficits in locomotor function and sensation resulting from oxidative stress [3]. Due to the absence of therapeutic agents, rat and mouse models of SCI (induced by ischemia, compression, contusion, and transection) are often used in the laboratory to develop innovative therapies. Melatonin [4], high-dose methylprednisolone [5], a Rho inhibitor [6], and riluzole [7] are currently being tested in humans and animals, but the efficacy of these agents is still the subject of debate. Therapeutic effects are often observed in laboratory animals but not in humans, which suggests that the specific functional mechanism of a drug rather than the drug itself is important. Usually, in addition to being efficacious in humans, drugs should have limited side effects and acceptable costs. Additionally, some agents remain in the animal experiment stage of development. In this context, an increasing number of novel drugs for SCI are emerging from the laboratory.

Many studies have shown that mesenchymal stem cells (MSCs) are promising cell therapy agents for both humans and animals with SCI, possibly through inhibition of inflammatory cascades [8,9,10]. As the product of stem cells, exosomes are considered to be important paracrine modulators and also the next generation of cell-free therapeutic agents for humans with SCI [11,12]. Exosomes are nano-sized 20- to 150-nm-diameter particles composed of a lipid bilayer that wraps RNA, DNA, and soluble proteins [13,14]. Due to their lipid bilayers, exosomes freely move through the blood, are absorbed by target cells, and can even pass through the blood–brain barrier.

Despite the unlikelihood of complete recovery, more researchers are acknowledging that exosomes can provide satisfactory improvements in motor function for exosomes. To determine whether exosomes are neuroprotective in rodent models of SCI, a systematic review of the efficacy of exosomes for the treatment of SCI is needed. Thus, we performed a systematic review and meta-analysis of data from studies investigating rodent models of SCI to assess the efficacy of exosomes for acute traumatic SCI.

2 Methods

2.1 Search strategies

This meta-analysis was limited to published articles on rodents and was performed by searching PubMed, EMBASE, Web of Science, Medline, Scopus, and the Cochrane Library databases (from inception to 2021). The search strategy is as follows: ((exosomes[title/abstract]) OR (extracellular vesicles[title/abstract]) OR (nano-sized vesicles[title/abstract]) OR (micro-vesicles)) AND (SCI[title/abstract]). The reference lists of the included articles were also searched to identify other studies. To perform a comprehensive search, we did not limit the “species”; articles reporting an unexpected “species” were excluded from the study selection process. A detailed database search strategy is provided in Table S1.

2.2 Study selection

All studies were stored as bibliographic references in NoteExpress (Aegean Sea Software Company, Beijing, China) and selected by two independent researchers (YW and XWL) based on the inclusion criteria. After primary selection, all articles were downloaded, and the articles that did not meet the inclusion criteria were excluded by browsing the specific content. A debate was resolved in consultation with a third investigator (HXY).

2.3 Eligibility criteria

The processing of articles followed the PICOS principle.

Type of participants (P): All studies included laboratory rats and mice subjected to acute SCI. Studies using nonmechanical methods such as radiation, electricity, and biochemical substances were excluded from the analysis.

Type of intervention (I): Studies that compared exosome administration to PBS, saline, or culture supernatant administration were included regardless of administration frequency, administration mode, and origin of the exosome.

Type of control (C): Studies with at least two intervention arms, with animals in the control group receiving placebo and animals in the experimental group receiving exosome administration, were included in this analysis.

Type of outcome (O): Studies that evaluated the locomotor function of the hind limbs of rats with the Basso, Beattie & Bresnahan (BBB) scale rather than the Basso Mouse Scale (BMS) and those that evaluated the locomotor function of the hind limbs of mice with the BMS rather than the BBB scale were included.

Type of study (S): All studies assessing the locomotor function recovery of SCI mice and rats were included.

2.4 Data extraction and quality assessment

Two skilled researchers (YW and XWL) independently extracted data from all articles meeting the inclusion criteria. The following data were extracted from the included studies: author, year, species, weight, the damaged segment of the spinal cord, anesthetic, SCI model, origin of exosomes, dose, administration frequency, and administration mode. When the data were presented as figures rather than tables, GetData Graph Digitizer 2.25 (Fedorov) was used to obtain the data. Based on our observations, the first analysis of mice and rats was usually conducted within 48 h, which may explain why the scores were presented as 0; in such cases, this measurement was not considered the first measurement. The quality of all included studies was evaluated by SYRCLE’s tool.

2.5 Outcome measurements

Behavioral improvement was assessed and recorded using the BBB locomotor rating scale for hind limb motor function in rats. The BBB scale, which ranges from 0 (no hind limb movement) to 21 (normal locomotion), was used to analyze specific improvements in locomotor function. The BMS, which ranges from 0 (no hind limb movement) to 9 (normal locomotion), was also used to assess motor function in mice. The movements of the hip, knee, and ankle joints were recorded when animals were allowed to move freely in an open field for 5 min.

2.6 Statistical analysis

The data from all included studies were summarized and analyzed by using R software version 3.6.3 (University of Auckland, New Zealand) and meta-package. All results reported in this review are presented as standardized mean differences (SMDs) with 95% CIs for outcomes. A random-effect model was used to analyze the data when heterogeneity was significant (p ≤ 0.05 or I 2 > 50%); otherwise, a fixed-effect model was used. Publication bias was tested by Egger’s t-test with R software version 3.6.3 and is presented as a funnel plot. Subgroup analyses of different models of SCI, administration modes, and measurement time points were also conducted. Trial sequential analysis (TSA) was conducted by using TSA software.

3 Results

The studies included in this meta-analysis were reported according to the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) (Table S2) [15].

3.1 Article selection process

The article selection process is shown in Figure 1. A total of 263 unique titles were retrieved from the databases. After removing duplicates and browsing the abstracts, 47 articles entered the full-text screening process. In this process, 1 article was excluded due to a lack of access. Following the full-text screening process, 11 studies, including one study that was withdrawn for plagiarism, two studies that used a different rating scale, one that utilized a rabbit model of SCI, and seven other studies that aim to investigate the pathophysiological development of SCI rats, were excluded. Ultimately, 35 articles, including two articles published in Chinese and 33 published in English, fully met the inclusion criteria set by the researchers.

Figure 1 
                  Summary of the article selection process.
Figure 1

Summary of the article selection process.

3.2 Study characteristics

As we investigated improvements in the motor function of both rats and mice, data for the two species were collected separately (Table 1). To collect as many critical factors as possible, the sample size of each group was not considered but is provided in the following figures and tables. Functional improvements in rats were reported in 29 studies, whereas the remaining six studies reported improved outcomes in mice.

Table 1

Characteristics of included studies reporting rats and mice

Author Year Species Gender Weight Segment Anesthesia Model Origin of exosome Dose Timing of injection Administration mode
Rat
Huang 2017 SD rats Male 180–220 g T10 10% chloral hydrate (0.3 mL/kg) Strike, 8 g × 40 mm BMSC 100 μg 30 min PI Tail vein injection
Pei 2017 NA NA NA T10 10% chloral hydrate (0.33 mL/kg) Strike, 200 kilodyne BMSC 50 μL (200 μg/mL) 1 h PI Tail vein injection
Ruppert 2017 SD rats Male 225–250 g T10 1.5 L/min of 2–3% isoflurane Strike, 50 kdynes with 1 s dwell hUC-MSC 109 particles/mL (1 mL) 3 h PI Tail vein injection
Kang 2018 SD rats NA 180–220 g T9/10 10% chloral hydrate (0.33 mL/kg) Strike,10 g × 25 mm miR-21, or PTEN siRNA transfected BMSC NA NA Tail vein injection
Huang 2018 SD rats NA 180–220 g T10 10% chloral hydrate (3 mg/kg) Strike, 8 g × 40 mm HUVEC, miR-126 transfected HUVEC 100 μg 30 min PI Tail vein injection
Jia 2018 SD rats Male 200–250 g T10 4% isoflurane and 2% isoflurane Strike, 200 kilodyne BMSC 200 μL (200 μg/mL) 30 min PI Tail vein injection
Li 2018 SD rats Male 250–300 g T10 Chloral hydrate (400 mg/kg) Compression,35 g × 60 s BMSC, miR-133b transfected BMSC 100 μg 24 h PI Tail vein injection
Liu 2018 SD rats Female 170–220 g T10 Chloral hydrate (350 mg/kg) Strike, 10 g × 12.5 mm BMSC 200 μg Immediately Tail vein injection
Tsai 2018 SD rats Female NA T9 NA Strike, 10 g × 5 mm BMSC NA 1, 2, and 3 DPI Tail vein injection
Wang-1 2018 SD rats NA 200–250 g T10 4% isoflurane and 2% isoflurane Strike, 200 kilodyne BMSC 200 μL (200 μg/mL) 30 min PI Tail vein injection
Wang-2 2018 SD rats Male 200–250 g T10 4% isoflurane and 2% isoflurane Strike, 200 kilodyne BMSC 100 μL (200 μg/mL) 2 h PI (every other day subsequently) Tail vein injection
Xu 2018 SD rats NA 180–220 g T9/T10 10% chloral hydrate (0.33 mL/kg) Strike, 10 g × 25 mm Undifferentiated PC12 cell, differentiated PC12 cell NA NA Tail vein injection
Ji 2019 SD rats Male 150–180 g T10 60 mg/kg sodium pentobarbital Compression, 35 g × 60 s BMSC 100 mg 24 h PI Tail vein injection
Guo 2019 SD rats Male 200–250 g T10 1–2% isoflurane, ketamine (60–90 mg/kg) and xylazine (10–15 mg/kg) Complete transection BMSC 40 μL 2–3 h postoperation; every other 24 h for 5 days Intrathecal injection
Huang 2019 SD rats Male 180–220 g T10 60 mg/kg ketamine and 6 mg/kg xylazine Strike, 8 g × 40 mm EF‑MSC 100 μg Immediately Intrathecal injection
Rong-1 2019 SD rats Male 180–220 g T10 50 mg/kg pentobarbital Strike, 10 × 12.5 mm NSC 200 µg Immediately Tail vein injection
Rong-2 2019 SD rats Male 180–220 g T10 NA Strike, 10 g × 12.5 mm NSC 200 μg Immediately Tail vein injection
Wang 2019 SD rats NA 180–220 g T9/T10 10% chloral hydrate (0.3 mL/kg) Strike, 10 g × 25 mm PTEN siRN and miR-21/miR-19a transfected PC12 cells NA NA NA
Yu 2019 SD rats Female 230–250 g T10 1% pentobarbital (80 mg/kg) Strike, 200 kilodyne BMSC 200 μg/mL 1 h PI Tail vein injection
Zhao 2019 Wistar rats Male 200–250 g T10 4% isoflurane, 2% isoflurane Compression BMSC 500 µL/min 1 h PI Tail vein injection
Zhou 2019 Wistar rats Male 200–250 g T10 2% isoflurane, 0.8 % isoflurane Transection BMSC 100 μg 1 h PI Tail vein injection
Li 2019 Wistar rats Male 150–200 g T9–T11 10% chloral hydrate (0.33 mL/kg) Strike, 10 g × 5 cm BMSC 200 μg Immediately NA
Guo 2020 SD rats Male 220–260 g T10 10% chloral hydrate (3 mL/kg) Strike, 10 g × 12.5 mm BMSC 1 μg/μL 1 h PI Tail vein injection
Kang 2020 SD rats Male 180–220 g T9/T10 10% chloral hydrate (0.33 mL/kg) Strike, 10 g × 25 mm miR-29b transfected PC12 cells NA NA Tail vein injection
Li-1 2020 SD rats Male NA L2–L5 10% chloral hydrate (3 mL/kg) Ischemia BMSC 5 × 1010 particles/100 μL NA Tail vein injection
Li-2 2020 SD rats Male NA T10 10% chloral hydrate (3 mL/kg) Compression, 20 s BMSC 100 μg 24 h PI Tail vein injection
Li-3 2020 NA NA NA NA NA NA MSC NA NA NA
Luo 2020 SD rats Male 170–220 g T10 1% pentobarbital sodium (40 mg/kg) Strike, 10 g × 12.5 mm BMSC 200 μg Immediately Tail vein injection
Moham med 2020 Wistar rats Male 250–300 g T9 and T10 80 mg/kg ketamine and 15 mg/kg xylazine Compression, 50 g × 5 min NSC 10 μg NA Intrathecal injection
Mouse
Liu 2020 C57BL/6 Male 6–8 W T10 Halothane Contusion, 5 g × 6.5 cm BMSC 200 μL Immediately Tail vein injection
Sun 2018 C57BL/6 Female 17–22 g T11/T12 50 mg/kg pentobarbital 10 g × 6.25 mm hUC-MSC 1 μg/mL 30 min PI Tail vein injection
Wang 2020 C57BL/6 J Female 18–22 g T10 2.0% isoflurane Strike, 5 g × 5 cm BMDM 200 μL 30 min PI Tail vein injection
Zhong 2020 C57BL/6 Female 25–30 g T10 Pentobarbital sodium Strike, 8 g × 3 cm NSC 200 μg 30 min PI Tail vein injection
Yuan 2019 ICR Male 8 W T10 1.5% isoflurane Strike, 50 kilodyne 20 μg 3, 6, 9, and 12 DPI Tail vein injection
Shao 2020 C57BL/6 NA 6 W T8/T9 30 mg/kg pentobarbital sodium Compression, depth of 0.2 mm × 20 s SCMEC 200 μg 1 h PI Tail vein injection

SD, Sprague–Dawley; MSC, mesenchymal stem cell; HUVEC, human umbilical venous endothelial cell; NSC, neural stem cell; EF‑MSC, epidural fat mesenchymal stem cell; NA, not available; BMSC, bone marrow-derived stem cell; ICR, institute of cancer research; SCEMC, spinal cord microvascular endothelial cell; NSC, neural stem cells; BMDM, bone marrow-derived macrophage; DPI, day post injury; PI, post injury; hUC-MSC, human unbilical cord mesenchymal stem cell; W, week; T, thoracic; h, hour; mg, milligram; kg, kilogram; mm, millimete; cm, centimeter; s, second; min, minute.

Of the studies on rat models of SCI, one trial used an ischemic model, two trials used a transection model, three trials utilized clip compression, and the remaining trials used Allen’s model or an Infinite Horizon impactor providing a force of 200 kilodynes. Of the studies on mouse models, one used an SCI model of compression, and the rest utilized Allen’s model. Male rats and female mice were the preferred rodent models of SCI. The dose of exosomes applied in these experiments ranged from 10 to 200 μg; however, it was difficult to attain dosing information, and some trials reported only the concentration of exosomes. Exosomes were mainly injected via the tail vein and subarachnoid space within 24 h. The analyzed studies used exosomes that originated from MSCs, including bone marrow MSCs, human umbilical cord MSCs, adipose-derived MSCs, human umbilical vein endothelial cells (HUVECs), and rat pheochromocytoma (PC12) cells, as well as other cell types.

3.3 Comparison of BBB scores between exosome-treated and control rats

We analyzed all studies (n = 583 animals) reporting locomotor recovery in rats at the first measurement. BBB scores reflecting the movement level of the hind limbs of exosome-treated rats were slightly but significantly improved (0.61, 95% CI: 0.21–1.01, p < 0.01) compared to those of rats in the control group at the first measurement (Figure 2). Furthermore, the data collected from the last measurement (3.21, 955 CI: 2.68–3.73, p < 0.01), which were reported in 29 studies, and the pooled analysis showed a similar outcome (Figure 3).

Figure 2 
                  Pooled-analysis of Basso, Beattie, and Bresnahan scale at the first measurement after SCI. SCI, spinal cord injury; SMD, standard mean difference; SD, standard difference; CI, confidential interval.
Figure 2

Pooled-analysis of Basso, Beattie, and Bresnahan scale at the first measurement after SCI. SCI, spinal cord injury; SMD, standard mean difference; SD, standard difference; CI, confidential interval.

Figure 3 
                  Pooled-analysis of Basso, Beattie, and Bresnahan scale at the last measurement after SCI. SMD, standard mean difference; SD, standard difference; CI, confidential interval.
Figure 3

Pooled-analysis of Basso, Beattie, and Bresnahan scale at the last measurement after SCI. SMD, standard mean difference; SD, standard difference; CI, confidential interval.

3.4 Comparison of BMS scores between exosome-treated and control mice

Six of the studies (n = 116 animals) evaluated the effect of exosomes on locomotor function. No remarkable improvements in the mice that received exosome administration compared to mice that received placebo administration were observed at the first measurement (0.48, 95% CI: −1.01 to 1.97, p < 0.01) (Figure 4a). At the last measurement, compared to placebo, exosomes increased the locomotor function of mice (2.46, 95% CI: 1.20–3.72, p < 0.01) (Figure 4b).

Figure 4 
                  Pooled-analysis of Basso Mouse scale at the first (a) and last measurement (b) after SCI. SMD, standard mean difference; SD, standard difference; CI, confidential interval.
Figure 4

Pooled-analysis of Basso Mouse scale at the first (a) and last measurement (b) after SCI. SMD, standard mean difference; SD, standard difference; CI, confidential interval.

3.5 Trial sequential analysis

TSAs were performed for rats and mice at the end of the follow-up day in a random-effects model meta-analysis with an overall significance level (α) of 0.05 and a type II error risk (β) of 0.1 (i.e., power 90%) preset (Figure 5). The cumulative Z-curve for rats crossed the upper monitoring boundary for the benefit and the adjusted required information size was calculated as 71 accrued rats, confirming a beneficial effect of exosomes on locomotor recovery (Figure 5a). Similarly, the TSA proved the beneficial effect of exosomes on locomotor recovery in SCI mice and the adjusted information size was calculated as 46 accrued mice (Figure 5b).

Figure 5 
                  TSAs of the effect of exosomes on locomotor recovery after SCI. (a) The adjusted required information size is based on a median value of mean BBB scores of 3.21, an overall significance level (α) of 0.05, a type II risk (β) of 0.1 (power 90%), and equals 71 rats (vertical dotted red line). The cumulative Z-curve (solid blue line) connected by individual studies (small squares) crosses the upper O’Brien–Fleming monitoring boundary of benefit (descending dotted red line). (b) The adjusted required information size is based on a median value of mean BBB scores of 2.46, an overall significance level (α) of 0.05, a type II risk (β) of 0.1 (power 90%), and equals 46 rats (vertical dotted red line). The cumulative Z-curve (solid blue line) connected by individual studies (small squares) crosses the upper O’Brien–Fleming monitoring boundary of benefit (descending dotted red line).
Figure 5

TSAs of the effect of exosomes on locomotor recovery after SCI. (a) The adjusted required information size is based on a median value of mean BBB scores of 3.21, an overall significance level (α) of 0.05, a type II risk (β) of 0.1 (power 90%), and equals 71 rats (vertical dotted red line). The cumulative Z-curve (solid blue line) connected by individual studies (small squares) crosses the upper O’Brien–Fleming monitoring boundary of benefit (descending dotted red line). (b) The adjusted required information size is based on a median value of mean BBB scores of 2.46, an overall significance level (α) of 0.05, a type II risk (β) of 0.1 (power 90%), and equals 46 rats (vertical dotted red line). The cumulative Z-curve (solid blue line) connected by individual studies (small squares) crosses the upper O’Brien–Fleming monitoring boundary of benefit (descending dotted red line).

3.6 Locomotor function recovery of rats and mice on the 3rd, 7th, 14th, 21st, and 28th day post injury

Most studies continuously measured the BBB scores of rats on the 3rd, 7th, 14th, 21st, and 28th day post injury (DPI; Figure 6). On the 3rd (0.65, 95% CI: 0.19–1.11, p < 0.01), 7th (1.92, 95% CI: 1.48–2.36, p < 0.01), 14th (2.70, 95% CI: 1.48–2.36, p < 0.01), 21st (3.29, 95% CI: 2.65–3.94, p < 0.01), and 28th (3.38, 95% CI: 2.71–4.05, p < 0.01) DPI, great improvements in locomotor function were observed in rats. Furthermore, we found that, over time, the difference prominently increased.

Figure 6 
                  Locomotor function recovery of mice and rats on the 3rd, 7th, 14th, 21st, and 28th DPI. BBB, Basso, Beattie, and Bresnahan; BMS, Basso Mouse scale; DPI, day post injury; SMD, standard mean difference.
Figure 6

Locomotor function recovery of mice and rats on the 3rd, 7th, 14th, 21st, and 28th DPI. BBB, Basso, Beattie, and Bresnahan; BMS, Basso Mouse scale; DPI, day post injury; SMD, standard mean difference.

Meanwhile, exosome-treated mice exhibited similar improvements in locomotor function on the 3rd (1.33, 95% CI: 0.01–2.64, p < 0.01), 7th (2.01, 95% CI: 0.72–3.30, p < 0.01), 14th (3.08, 95% CI: 2.11–4.06, p < 0.01), 21st (1.98, 95% CI: 0.09–3.88, p < 0.01), and 28th (3.44, 95% CI: 2.12–4.76, p < 0.01) DPI. Over time, mice that received exosomes injection exhibited increasingly higher BMS scores than mice that received placebo injection (Figure 6).

3.7 Subgroup analysis

Four kinds of rat models of SCI (ischemia, compression, contusion, and transection) were used, and we conducted subgroup analyses of data from different rat models of SCI. The ischemic model was not subjected to subgroup analysis due to the limited number of articles that used this model (n = 1).

Great improvements in BBB scores were observed in contusion models (0.74, 95% CI: 0.03–1.45, p = 0.04), but no improvements in BBB scores were observed in the compression models (−1.25, 95% CI: −4.01 to 1.52, p = 0.38) at 3rd DPI; this suggested that rats in compression model trended to recover slower than rats in contusion model. On average, rats in the transection model seemed to get a higher SMD value than rats in contusion and compression models; however, this point should be cautiously concluded owing to the lack of direct evidence (Table 2).

Table 2

Subgroup analysis of rat models, administration modes and exosome origins

Subgroup No. of rats (Exo) No. of rats (SCI) SMD 95% CI p value
SCI model
Contusion
3d 17 17 0.74 [0.03–1.45] 0.04
7d 38 32 2.08 [0.92–3.24] 0.03
14d 38 32 3.19 [1.08–5.31] <0.01
21d 32 26 4.79 [0.06–8.98] 0.03
28d 32 26 3.76 [1.56–5.97] <0.01
Compression
3d 41 31 −1.25 [−4.01 to 1.52] 0.38
7d 41 31 2.00 [0.59–3.40] <0.01
14d 21 21 1.87 [1.10–2.64] <0.01
21d 15 15 2.24 [−0.10 to 4.58] 0.06
28d 15 15 3.05 [0.50–5.61] 0.03
Transection
7d 27 21 3.06 [2.17–3.95] <0.01
14d 27 21 5.19 [3.92–6.46] <0.01
21d 27 21 5.83 [4.43–7.23] <0.01
28d 27 21 4.87 [3.66–6.08] <0.01
Administration
Tail vein injection
3d 220 164 0.38 [−0.10 to 0.85] 0.12
7d 235 170 1.69 [1.17–2.21] <0.01
14d 221 166 2.53 [1.97–3.08] <0.01
21d 153 199 2.81 [2.14–3.47] <0.01
28d 199 157 2.84 [2.21–3.48] <0.01
Intrathecal injection
3d 18 26 0.69 [0.06–1.32] 0.03
7d 39 41 2.22 [0.96–3.48] <0.01
14d 39 41 4.05 [2.14–5.95] <0.01
21d 39 41 6.26 [3.12–9.39] <0.01
28d 39 41 5.86 [3.55–8.16] <0.01
Exosome origine
BMSC
3d 149 130 0.15 [−0.48 to 0.77] 0.65
7d 188 154 1.58 [0.84–2.33] <0.01
14d 188 154 3.04 [2.38–3.71] <0.01
21d 133 124 3.09 [2.22–3.96] <0.01
28d 168 144 3.11 [2.32–3.89] <0.01
Gene-modified BMSC
3d 12 9 0.57 [–0.36 to 1.49] 0.23
7d 12 9 3.11 [–1.20 to 7.42] 0.16
14d 12 9 1.33 [0.32–2.34] 0.01
NSC
3d 30 22 0.91 [0.32–1.50] <0.01
7d 30 22 2.31 [0.47–4.14] <0.01
14d 30 22 2.91 [1.39–4.44] <0.01
21d 30 22 3.68 [1.83–5.53] <0.01
28d 30 22 3.81 [1.72–5.91] <0.01
PC12
3d 25 14 1.51 [0.20–2.81] 0.02
7d 25 14 1.26 [0.51–2.02] <0.01
14d 25 14 0.93 [0.20–1.66] 0.01
21d 25 14 2.35 [1.43–3.26] <0.01
28d 25 14 3.07 [2.00–4.13] <0.01

SMD, standard mean difference; Exo, exosomes; SCI, spinal cord injury; CI, confidential intervals; PC12, pheochromocytoma; BMSC, bone marrow-derived mesenchymal stem cell.

Note: That bold italic value indicates that the SMD value between Exo and SCI group is non-significant.

Among included articles, intrathecal and tail vein injections were mainly utilized. Our subgroup analysis seemed to prefer intrathecal injection because the significant promotion of locomotor function in rats receiving tail vein injection was not observed at the 3rd DPI (0.38, 95% CI: −0.10 to 0.85, p = 0.12) to the 7th DPI (1.69, 95% CI: 1.17–2.21, p < 0.01); however, rats receiving intrathecal injection had already got significant locomotory function recovery at the 3rd DPI (0.69, 95% CI:0.06–1.32, p = 0.03) (Table 2).

Subsequently, we analyzed the effect of exosomes from bone marrow-derived mesenchymal stem cell (BMSC), gene-modified BMSC, neuronal stem cell (NSC), and PC12 cells. All exosomes showed satisfying therapeutic effects on SCI. However, exosomes from NSC (3rd DPI, 0.91, 95% CI: 0.32–1.50, p < 0.01) and PC12 cells (3rd DPI, 1.51, 95% CI: 0.20–2.81, p < 0.01) seemed to take effect earlier than exosomes from BMSC (3rd DPI, 0.15, 95% CI: −0.48 to 0.77, p = 0.65) and gene-modified BMSC (3rd DPI, 0.57, 95% CI: −0.36 to 1.49, p = 0.23) (Table 2). Finally, we also determined that species, year, gender, and injured segment of the spinal cord were not sources of heterogeneity by using meta-regression.

3.8 Bias risk

We evaluated the article quality using SYRCLE’s tool (Table 3). The results showed that most articles reported randomness and blindness, and the rest articles reported either randomness or blindness. Other bias indexes were low. Publication biases for BBB scores at the first measurement (Figure S1a; Egger’s test, p = 0.907), BBB scores at the last measurement ((Figure S1b; Egger’s test, p = 0.00), BMS scores at the first measurement ((Figure S1c; Egger’s test, p = 0.767) and BMS scores at the last measurement (Figure S1d; Egger’s test, p = 0.066) were tested by funnel plots and Egger’s linear regression.

Table 3

Article quality assessment using SYRCLE’s tool

Author/Year Random sequence Allocation concealment Baseline characteristics Blinding (Study team) Random housing Random outcome assessment Blinding (Outcome assessors) Incomplete outcome data Selective outcome reporting
Selection bias Detection bias Reporting bias Attrition bias Reporting bias Other bias
Rat
Huang/2017 + ? + + + + +
Pei/2017 + ? ? ? ? ? + + +
Ruppert/2017 + ? + ? + + + + + +
Kang/2018 + ? + ? + ? + + +
Huang/2018 + ? + + ? + + + +
Jia/2018 + + ? + ? + + + +
Li/2018 + + ? + ? + + + +
Liu/2018 + + + + + ? + + + +
Tsai/2018 ? + ? + + + +
Wang-1/2018 + + + + ? + + + +
Wang-2/2018 + ? ? + + + +
Xu/2018 + ? + + ? + + +
Ji/2019 + ? + + +
Guo/2019 ? + + ? ? + + +
Huang/2019 + ? + + + ? ? + + +
Rong-1/2019 + ? + + + ? + + + +
Rong-2/2019 + ? + + + ? + + + +
Wang/2019 + ? ? + + + + +
Yu/2019 + + ? + ? + + +
Zhao/2019 + + + + ? ? + + +
Zhou/2019 + ? + + + ? + + + +
Li/2019 + ? + + + ? ? + + +
Guo/2020 + ? + ? ? ? + + +
Kang/2020 + ? + ? ? + + +
Li-1/2020 ? ? + ? ? + + +
Li-2/2020 + ? + + + ? + + + +
Li-3/2020 + ? ? + ? + + +
Luo/2020 ? + + ? + + + + +
Mohammed/2020 + ? + + ? ? + + +
Mouse
Wei/2020 ? + + + + + + + +
Sun/2018 ? ? + + + ? + + + +
Wang/2020 ? ? + ? ? ? + + +
Zhong/2020 ? ? + ? ? ? + + +
Shao/2020 ? ? + + ? + + + + +
Yuan/2019 ? ? + + + ? + + + +

(+) low risk of bias; (−) high risk of bias; (?) unclear risk of bias.

4 Discussion

To ensure reproducibility from the laboratory to the clinic, stringent animal studies should be performed, and the molecular mechanisms involved in neuroprotection should be identified. Herein, we conducted a meta-analysis of all accessible articles to assess the potential clinical translation of exosomes.

4.1 Summary of the evidence

This meta-analysis included 35 articles involving 699 rodents (rat, n = 583; mouse, n = 116) and compared the effects of exosomes with those of placebo. Differences of pooled analysis in the recovery of motor function of rats and mice were identified. Subgroup analysis revealed that the differences between exosome- and placebo-treated animals became greater over time. Rats in the compression model trended to recover more slowly than rats in contusion and transection models. Moreover, rats treated by intrathecal injection seemed to recover faster than rats treated by tail vein injection; however, this conclusion needs to be verified by more studies due to the lack of direct comparison. Many previous studies have reported distinct promotion of locomotor function recovery on the 7th DPI, but our findings seem to report earlier recovery on the 3rd day in rats, which is promising. Furthermore, because different rating scales were used, we should be cautious in concluding that rats recover from SCI more quickly than mice; this point should be addressed in future studies.

Rating scale (e.g., BBB and BMS) is a relatively subjective tool, especially while the score is recorded by different performers. We recommend more objective tools, such as the force of the hind limbs, motor-evoked potential (MEP), and sensory-evoked potential (SEP) while evaluating the locomotor function. Additionally, as for the experimental model for SCI, the researchers have not reached a consensus. The establishment of a standardized and globally accepted SCI model should be on the way.

As evidenced by our results, the administration method merely impacts the onset time rather than the final therapeutic effect. Thus, the tail vein injection that potentially averts secondary damage to the spinal cord is more recommended.

4.2 Strengths and weaknesses

To the best of our knowledge, we are the first to perform a quantitative meta-analysis assessing the curative effect of exosomes on locomotor function recovery. We carefully considered the potential origins of heterogeneity encountered in future trials, such as dose, the timing of administration and administration method, which may contribute to future clinical translation.

Limitations of this study should be addressed. We found that most studies reported positive results; hence, we hypothesized that negative results were concealed and unpublished, resulting in potential bias and misleading results. As animal trials differ from randomized clinical trials (RCTs), it is difficult to collect the characteristics of each group in animal trials, and some critical data (SCI, model dose, and administration method) were missing from these original articles. Additionally, confusing information was sometimes reported; for example, some studies provided only the volume or concentration of exosomes, and four articles did not report the injured segment of the spinal cord. Owing to the small sample size, we should be cautious to conclude the locomotor function recovery in mice. Finally, the interpretation of observations depends heavily on the individual observer and whether the observer is blinded to the treatment group. Therefore, the efficacious translation of our results should be cautious.

4.3 Possible mechanism of exosomes

Trauma at the lesion site directly leads to apoptosis of neurons [16], activation of cells that support neurons [11] and subsequent activation of neurotoxic signaling cascades [17] in neuronal cells. Secondary damage (mainly inflammation) triggered by microglia, astrocytes, and other immune cells, cell death, and scar formation usually occur minutes to months after SCI [18]. Currently, it is gradually acknowledged that the promotion of neuron regeneration [19], inhibition of glial activation [20], and suppression of cell death by exosomes are closely intimately with the locomotor function recovery [11]. But the steps toward inner mechanisms should never cease.

4.4 Implications for future studies

Animal studies are important for translation to clinical trials and evaluation of interventions for clinical trials. Identification of phenotypes, which is an important step in drug development and research, is always first performed in animals, and the mechanisms of action are later identified. Despite the large amount of evidence proving that exosomes improve the locomotor function of SCI rats [21,22,23,24], many studies have only reported that exosome administration inhibits inflammation [11,12], which is not sufficient to support a clinical trial. The complex nature of exosomes results from their components and origins. Thus, more studies investigating the mechanisms involved in neural outgrowth, inactivation of microglia and astrocytes, and inhibition of cell apoptosis should be implemented to identify the mechanism by which the greatest effects are exerted.

5 Conclusion

The present meta-analysis suggested that exosomes improve the locomotor function of rodents with SCI, although the mechanism of action remains investigated.

However, the SCI model, administration method, and origin of exosome are potential factors of the therapeutic effect. Our findings should be interpreted with caution considering the disparity between species and provide some insights into future studies rather than definitive clinical recommendations.


Hanxiao Yi and Yang Wang contributed equally to this article.


  1. Funding Information: This work was supported by Nurturing funds for nursing young talents of Sun Yat-sen University (No. N2020Y06).

  2. Author contributions: Y.W. first presented the idea and designed the outline of the article. Both Y.W. and H.X.Y. were responsible for all data extraction and analysis. The first version was written by H.X.Y. The final version was revised by Y.W. Both Y.W. and H.X.Y. were responsible for the final submission.

  3. Conflict of interest: There are no conflicts of interests declared by all authors.

  4. Data availability statement: All data generated or analyzed during this study are included in this published article [and its supplementary information files].

Appendix

Figure S1 
Funnel plots were conducted for pooled analysis of BBB at the first measurement (a), pooled analysis of BBB at the last measurement (b), pooled analysis of BMS at the first measurement (c), and pooled-analysis of BMS the first measurement (d).
Figure S1

Funnel plots were conducted for pooled analysis of BBB at the first measurement (a), pooled analysis of BBB at the last measurement (b), pooled analysis of BMS at the first measurement (c), and pooled-analysis of BMS the first measurement (d).

Table S1

Search strategy and databases

Database Search strategy
PubMed ((exosomes)OR(extracellular vesicles)OR(nano-sized vesicles)OR(micro-vesicles))AND(spinal cord injury)
EMBASE
Wed of science
Medline
Scopus
Cochrane library
Table S2

PRISMA 2009 checklist

Section/topic # Checklist item Reported on page #
TITLE
Title 1 Identify the report as a systematic review, meta-analysis, or both. 1
ABSTRACT
Structured summary 2 Provide a structured summary including, as applicable: background; objectives; data sources; study eligibility criteria, participants, and interventions; study appraisal and synthesis methods; results; limitations; conclusions and implications of key findings; systematic review registration number. 2–3
INTRODUCTION
Rationale 3 Describe the rationale for the review in the context of what is already known. 3–4
Objectives 4 Provide an explicit statement of questions being addressed with reference to participants, interventions, comparisons, outcomes, and study design (PICOS). 4
METHODS
Protocol and registration 5 Indicate if a review protocol exists, if and where it can be accessed (e.g., Web address), and, if available, provide registration information including registration number. NA
Eligibility criteria 6 Specify study characteristics (e.g., PICOS, length of follow-up) and report characteristics (e.g., years considered, language, publication status) used as criteria for eligibility, giving rationale. 5–6
Information sources 7 Describe all information sources (e.g., databases with dates of coverage, contact with study authors to identify additional studies) in the search and date last searched. 5
Search 8 Present full electronic search strategy for at least one database, including any limits used, such that it could be repeated. 5
Study selection 9 State the process for selecting studies (i.e., screening, eligibility, included in systematic review, and, if applicable, included in the meta-analysis). 5
Data collection process 10 Describe method of data extraction from reports (e.g., piloted forms, independently, in duplicate) and any processes for obtaining and confirming data from investigators. 6–7
Data items 11 List and define all variables for which data were sought (e.g., PICOS, funding sources) and any assumptions and simplifications made. 6–7
Risk of bias in individual studies 12 Describe methods used for assessing risk of bias of individual studies (including specification of whether this was done at the study or outcome level), and how this information is to be used in any data synthesis. NA
Summary measures 13 State the principal summary measures (e.g., risk ratio, difference in means). 7–8
Synthesis of results 14 Describe the methods of handling data and combining results of studies, if done, including measures of consistency (e.g., I2) for each meta-analysis. 7–8
Risk of bias across studies 15 Specify any assessment of risk of bias that may affect the cumulative evidence (e.g., publication bias, selective reporting within studies). 7–8
Additional analyses 16 Describe methods of additional analyses (e.g., sensitivity or subgroup analyses, meta-regression), if done, indicating which were pre-specified. 8
RESULTS
Study selection 17 Give numbers of studies screened, assessed for eligibility, and included in the review, with reasons for exclusions at each stage, ideally with a flow diagram. 8
Study characteristics 18 For each study, present characteristics for which data were extracted (e.g., study size, PICOS, follow-up period) and provide the citations. 8–9
Risk of bias within studies 19 Present data on risk of bias of each study and, if available, any outcome level assessment (see item 12). NA
Results of individual studies 20 For all outcomes considered (benefits or harms), present, for each study: (a) simple summary data for each intervention group (b) effect estimates and confidence intervals, ideally with a forest plot. 9–13
Synthesis of results 21 Present results of each meta-analysis done, including confidence intervals and measures of consistency. 9–13
Risk of bias across studies 22 Present results of any assessment of risk of bias across studies (see Item 15). 12 to13
Additional analysis 23 Give results of additional analyses, if done (e.g., sensitivity or subgroup analyses, meta-regression [see Item 16]). 13
DISCUSSION
Summary of evidence 24 Summarize the main findings including the strength of evidence for each main outcome; consider their relevance to key groups (e.g., healthcare providers, users, and policy makers). 14 to15
Limitations 25 Discuss limitations at study and outcome level (e.g., risk of bias), and at review-level (e.g., incomplete retrieval of identified research, reporting bias). 15–16
Conclusions 26 Provide a general interpretation of the results in the context of other evidence, and implications for future research. 18–19
FUNDING
Funding 27 Describe sources of funding for the systematic review and other support (e.g., supply of data); role of funders for the systematic review. 19

From: Moher D, Liberati A, Tetzlaff J, Altman DG, The PRISMA Group (2009). Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement. PLoS Med 6(7): e1000097. 10.1371/journal.pmed1000097.

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Received: 2021-02-02
Revised: 2021-04-19
Accepted: 2021-05-06
Published Online: 2021-07-15

© 2021 Hanxiao Yi and Yang Wang, published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

Downloaded on 28.3.2024 from https://www.degruyter.com/document/doi/10.1515/med-2021-0304/html
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