Calpain inhibitor MDL28170 improves the transplantation-mediated therapeutic effect of bone marrow-derived mesenchymal stem cells following traumatic brain injury
- 267 Downloads
Studies have shown that transplantation of bone marrow-derived mesenchymal stem cells (BMSCs) protects against brain damage. However, the low survival number of transplanted BMSCs remains a pertinent challenge and can be attributed to the unfavorable microenvironment of the injured brain. It is well known that calpain activation plays a critical role in traumatic brain injury (TBI)-mediated inflammation and cell death; previous studies showed that inhibiting calpain activation is neuroprotective after TBI. Thus, we investigated whether preconditioning with the calpain inhibitor, MDL28170, could enhance the survival of BMSCs transplanted at 24 h post TBI to improve neurological function.
TBI rat model was induced by the weight-drop method, using the gravitational forces of a free falling weight to produce a focal brain injury. MDL28170 was injected intracranially at the lesion site at 30 min post TBI, and the secretion levels of neuroinflammatory factors were assessed 24 h later. BMSCs labeled with green fluorescent protein (GFP) were locally administrated into the lesion site of TBI rat brains at 24 h post TBI. Immunofluorescence and histopathology were performed to evaluate the BMSC survival and the TBI lesion volume. Modified neurological severity scores were chosen to evaluate the functional recovery. The potential mechanisms by which MDL28170 is involved in the regulation of inflammation signaling pathway and cell apoptosis were determined by western blot and immunofluorescence staining.
Overall, we found that a single dose of MDL28170 at acute phase of TBI improved the microenvironment by inhibiting the inflammation, facilitated the survival of grafted GFP-BMSCs, and reduced the grafted cell apoptosis, leading to the reduction of lesion cavity. Furthermore, a significant neurological function improvement was observed when BMSCs were transplanted into a MDL28170-preconditioned TBI brains compared with the one without MDL28170-precondition group.
Taken together, our data suggest that MDL28170 improves BMSC transplantation microenvironment and enhances the neurological function restoration after TBI via increased survival rate of BMSCs. We suggest that the calpain inhibitor, MDL28170, could be pursued as a new combination therapeutic strategy to advance the effects of transplanted BMSCs in cell-based regenerative medicine.
KeywordsTraumatic brain injury Bone marrow-derived mesenchymal stem cells Preconditioning Calpain inhibitor MDL28170 Transplantation
Analysis of variance
Bone marrow-derived mesenchymal stem cells
Enzyme-linked immunosorbent assay
Green fluorescent protein
Modified neurological severity score
Scanning electron microscope
Traumatic brain injury
Traumatic brain injury (TBI) remains a major health problem worldwide. The pathophysiology of brain injury after head trauma is complicated and can be characterized by the initial injury and the subsequent injury that ensues days after the trauma . The incidence of TBI is increasingly a major cause of morbidity and mortality among all traumas [2, 3], leading to considerable disability, mortality, and functional impairment that severely affects the quality of life [4, 5].
Currently, therapeutic strategies for TBI mainly include controlling the secondary damage through the administration of neurotrophic drugs and promoting rehabilitation training of neurological function . However, these therapeutic effects were less than optimal and novel strategies remain to be found. In the last decade, several studies regarding bone marrow-derived mesenchymal stem cell (BMSC) transplantation as an alternative therapy for TBI [7, 8, 9] have shown great promise in animal experimental models [10, 11, 12, 13] and in the clinic [14, 15]. The benefits of the transplanted BMSC are twofold: (i) its ability to commit to a neural lineage and migrate long distances to injury sites allows it to serve as a direct replacement for dead or dying cells [16, 17] and (ii) its presence at the lesion site indirectly influences the microenvironment through the secretion of growth factors, which rescues neuronal cells and promotes the proliferation of neuroblasts [18, 19]. Yet, the survival and viability of BMSCs are relatively poor in the injured brain, and the early death of transplanted cells limits the BMSC-based therapies [20, 21]. To exploit their full therapeutic potential, there is a critical need to determine the cause(s) of early death and develop strategies to enhance their survival.
Factors present at the lesion site can induce host tissue damage and contribute to the death of transplanted cells. Recent studies have demonstrated a pivotal role of calpain, a calcium-mediated cysteine protease, in mediating necrotic and apoptotic cell death . The resultant proteolysis of cytoskeletal, membrane, and myelin proteins is strongly implicated in the secondary damage, which includes the death of motor neurons, axonal degeneration, oligodendrocyte death, and demyelination-associated with Ca2+ accumulation . Meanwhile, the inflammatory response acts as a key step in the secondary injury cascade following TBI that also contributes to the death of transplanted cells. It is characterized by the recruitment of peripheral leukocytes into the cerebral parenchyma, activation of resident immune cells [24, 25], and initiation of the inflammatory cascade mediated by the release of pro- and anti-inflammatory cytokines [26, 27]. Several lines of evidence have highlighted calpain’s critical role in driving the inflammatory response, citing it as one of the earliest pro-inflammatory cytokines to be upregulated after neurotrauma [28, 29, 30].
Calpain modulates key processes that govern the pathogenesis of neurodegeneration and pro-inflammatory response [29, 31]. Therefore, calpain inhibitors can be presumed to be effective therapeutic agents for attenuating calpain’s actions [32, 33]. Here, we used the calpain inhibitor, MDL28170, which has the ability to cross the blood-brain barrier (BBB) and cell membranes. MDL28170 was reported to prevent the upregulation of pro-inflammatory factors induced by calpain [28, 34]. It also exerts neuroprotective effects in a variety of neurological injuries such as TBI, spinal cord injury, stroke, and Parkinson’s disease [31, 34, 35].
This study was designed to explore whether the anti-neurodegeneration and anti-inflammatory effects of the calpain inhibitor, MDL28170, could exert a certain protective effect against damage caused by TBI and enhance the survivability of grafted BMSCs in the contused rat brain to further improve the therapeutic effects of BMSC-based TBI therapy.
Experimental groups and TBI model
For the TBI model, rats were anesthetized by intraperitoneal injection with 10% chloral hydrate (0.4 mL/100 g), shaved, and placed in a stereotaxic frame (Kopf Instruments, Tujunga, CA, USA). The scalp was incised at the midline, exposing the skull. A right parietal bone was drilled with a hole of 5 mm in diameter without damaging the dura mater. The center of the craniotomy was 3.5 mm posterior and 2.5 mm lateral to the bregma. The parietal contusion was produced by allowing a 50 g hammer to fall from a 30-cm guide stick. At the end of the procedure, the exposed dura was covered with bone wax and the scalp was sutured. Sham-operated rats were surgically treated with right parietal craniotomy but without injury to the brain. After trauma, the rats were placed in a warmed, oxygenated recovery chamber with free under controlled temperature (25 ± 0.5 °C) and humidity (55 ± 5%). Rats were housed under the 12/12-h light-dark cycle and had unlimited access to food and water. Postoperative care included injections of penicillin to prevent infection. Rats that lacked neurological deficits after TBI administration were excluded.
MDL28170 (carbobenzoxy-valyl-phenylalanine, calpain inhibitor I, readily crosses the blood-brain barrier and cell membranes; Cat. No. M6690, Sigma, St Louis, USA)  was first dissolved in dimethylsulfoxide (DMSO) and then diluted with 0.9% NaCl to a final concentration of 50 mM. Final concentration of DMSO was 20%, v/v. At 30 min post TBI, 1.0 μl of 50 mM MDL28170 was injected into the center of the lesion site at a depth of 1.0 mm using a microinjection needle clamped by a stereotaxic instrument. Controls received an equal volume of the vehicle (20% DMSO, v/v).
Cell preparation, characterization and transplantation
Primary bone marrow stem cells were harvested from the bone marrow of SD rats, cultured as monolayer, then transfected with a lentiviral construct containing a green fluorescent protein (GFP) expression motif. GFP-BMSCs were cultured in a BMSC growth medium, passaged and amplified to the first generation, and frozen at − 80 °C. When needed, GFP-BMSCs were thawed and transferred to tubes containing the growth medium then centrifuged at 1000 rpm for 5 min. After removing the supernatant, cells were dispersed gently with 2–3 mL of medium. The cell suspension was transferred to a 25-cm2 flask, additional medium was added to reach a total volume of 4 ml and incubated in a carbon dioxide incubator (37 °C, 5% CO2). The medium was replaced every 3–4 days based on the rate of cell growth and the change in the color of the medium. To confirm the expression of GFP in BMSCs in vitro, we performed immunofluorescence staining using a GFP antibody (1:500, Santa Cruz Biotechnology), and cell nuclei were counterstained with DAPI (1:1000, Life Technologies). The GFP expression efficiency (%) was defined as the ratio of GFP-positive cells divided by the total number of cells (DAPI positive) per field. Five random fields per each well and four different wells at the same condition were evaluated to get the statistic value. Cell morphology was determined using a scanning electron microscope (SEM).
For GFP-BMSC transplantation treatment groups, cells were trypsinized with 0.05% trypsin solution for 3 min at 37 °C. After rinsing thrice, cells were used for transplantation. 1 × 105 cells in 3 μL of DMEM medium were engrafted into the epicenter of the injury site at a delivery rate of 1 μL/min with a microinjection needle. The total number of cells for each treatment was the same. Animals in other groups received only saline injections.
Enzyme-linked immunosorbent assay (ELISA)
To examine the inflammatory response at 24 h after MDL28170 treatment, brain tissue of the injected site was isolated and placed on ice. Each brain tissue was homogenized in RIPA lysis buffer (Thermo Fisher, USA) with the addition of protease inhibitors then centrifuged for 15 min at 12,000 rpm, 4 °C. The colorimetric ELISA kits were used to detect the cytokines (IL-1β, IL-6, TNF-α, IL-4, and IL-10) and transcription factor (NFκB) in the brain protein extract (R&D Systems, USA). For each ELISA analysis, 40 μL of sample was used without dilution in accordance with the manufacturer’s instructions.
Survival assay of grafted cells
Rats were anesthetized with a lethal dose of chloral hydrate and transcardially perfused with 100 mL of saline followed by 100 mL of 4% paraformaldehyde (PFA) in 0.1 M PBS (pH 7.6). The tissue was fixed overnight in 4% PFA in 0.1 M PBS at 4 °C and cryoprotected in 30% sucrose for 36 h. Frozen sections of 10 μm thickness were prepared and fixed in 4% PFA for 20 min, washed with PBS (5 min each time for three times), then permeabilized with 0.3% Triton X-100 for 15 min, and washed with PBS (5 min each time for three times). The transplanted BMSCs can be detected directly with the 488 nm wavelength due to the transfection of GFP; cell nuclei were counterstained with DAPI. Samples were analyzed by fluorescence microscopy (BX51, Olympus, Japan). Five microscopic fields (× 40) from each section of each rat in each BMSC transplantation group were acquired to perform subsequent statistical analyses.
Lesion volume assessment
Rats were sacrificed and transcardially perfused with saline and 4% PFA 7 days after cell transplantation. Sections were stained with Cresyl violet acetate, dehydrated, and mounted for analysis. The investigator measuring lesion area and contralateral hemisphere brain area using the NIH ImageJ program was blinded to the experimental conditions. Areas were multiplied by the distance between sections to obtain the respective volumes. Lesion volume was calculated as described previously : (lesion volume/volume of contralateral hemisphere) × 100%.
At 30 min post TBI, 1.0 μl of 50 mM MDL28170 was injected into the center of the lesion site at a depth of 1.0 mm using a microinjection needle clamped by a stereotaxic instrument. Controls received an equal volume of the vehicle (20% DMSO, v/v). At 24 h after TBI, the consistent cortex tissue region of TBI area was separated. The routine detail procedures of western blot have been showed previously . The following primary antibodies have been performed, including Bcl2 (Abacm, Rabbit, ab59348, 1:1000), Bax (Abacm, Rabbit, ab32503,1:1000), NFκB (Cell Signaling, Rabbit, #2144, 1:1000), p-IκB (Cell Signaling, Mouse, #2859, 1:1000), IκB (Cell Signaling, Rabbit, #4814, 1:1000), and α-tublin (Cell Signaling, Rabbit, #2144, 1:1000). For statistical analysis, each group contains three rats.
To explore the effect of MDL28170 on microgila activation, at 24 h after TBI, rats were sacrificed. The routine detail procedures for IbaI (Abcam, Goat, ab5076, 1:250) staining have been previously described . For statistical analysis, four random images around TBI area were taken from each slide and each group contains four rats.
Assessment of neurological function
Neurological function was assessed by a modified neurological severity score (mNSS) on the day before (baseline) and on days 7, 14, and 28 after transplantation by an investigator who was blinded to the experimental groups. The evaluations included motor, sensory, reflex, and balance tests. Neurological function was graded on a scale of 0–18 as previously described [38, 39]; the higher the score, the more severe the neurological impairment is. All rats were given enough time to become familiar with the testing environment before performing TBI, which was assessed by the rat’s ability to perform all the tests and a total mNSS (baseline) could be calculated.
The data are presented as mean ± standard deviation. All values were analyzed using Prism software (GraphPad, USA). To compare differences between two groups, unpaired Student’s t test was used. For comparing differences involving three or more groups, one-way or two-way analysis of variance (ANOVA) was utilized. A p value of less than 0.05 or 0.01 or 0.001 is considered statistically significant.
Characterization of cultured GFP-BMSCs
MDL28170 treatment in acute TBI phase decreased inflammatory effects
MDL28170 enhanced the survival ratio of grafted cells in host tissue
MDL28170 reduced lesion volume after transplantation of BMSCs in TBI
Assessment of neurological function after BMSC transplantation
MDL28170 reduced cell apoptosis and inhibited NFκb-Iκb signaling pathway after TBI
Previously, we have showed that the expression of NFκb after TBI was downregulated by MDL28170 treatment in the ELISA assay (Fig. 3d). This data agrees well with the western blot results, which also support that the protein level of NFκB was decreased after MDL28170 treatment (Fig. 7e, f). As we know, Iκb and p-Iκb are the downstream biomarkers of NFκB, and NFκB can mediate the phosphorylation of Iκb. Interestingly, our data showed that MDL28170 decreased the protein level of p-Iκb; however, no significant effect of protein level of Iκb was observed here (Fig. 7e, g, h). Put together, the results demonstrated that the administration of MDL28170 after TBI could inhibit cell apoptosis and reduce inflammation level by inhibit NFκB-Iκb signaling pathway.
MDL28170 administration inhibited microglia activation after TBI
In this study, our results demonstrate for the first time that the calpain inhibitor, MDL28170, administered by intracranial microinjection shortly following injury can not only attenuate the effects of an inflammatory microenvironment, but also enhance the survival rate of BMSCs at the contusive site, decrease lesion volume, and improve functional outcome. Taken together, our results provide preclinical experimental evidence for the efficacy of combinatorial therapy with MDL28170 and BMSCs to aid in functional recovery after a brain injury.
The effects of acute TBI include a complex cascade of pathophysiological sequelae such as excitotoxicity, generation of free radicals (elevated levels of reactive oxygen species and reactive nitric oxide), release of inflammatory molecules, and diffuse axonal and neuronal injury [40, 41]. Inflammatory responses are reported to be a crucial mechanism in secondary injury after TBI. Early responses of the inflammatory reactive cells result in a conspicuous accumulation of other inflammatory mediators such as cytokines and adhesion molecules [42, 43]. The massive death of donor cells in the contusion area during the acute phase resulting from increased free radicals and inflammatory responses immensely lowers the efficacy of the cell-based treatment. In order to improve the effect of stem cell-based therapy, various strategies have been adopted to develop and optimize the protocols to enhance donor stem cell survival post-transplantation, with special attention being paid to preconditioning approaches [44, 45]. Presently, several preconditioning triggers are being tested in stem cell-based therapy and have shown to increase the tolerance of transplanted cells to multiple injurious insults [46, 47].
An increasing number of studies suggest that calpains could participate in acute and chronic inflammatory processes under pathological conditions by acting as inflammatory regulators. For example, treatment with calpain inhibitor can reduce calpain activity in immune cells in the periphery to potentially block T cell activity and immune cell migration . In accordance with the literature, our study also showed that MDL28170 as a calpain inhibitor could alleviate the microglia activation at the lesion site of brain after TBI (Fig. 8). As reported recently, an increased calpain activity also correlates with greater production of pro-inflammatory IL-2/IFN-γ cytokines and decreased levels of anti-inflammatory cytokines IL-10 and IL-4, suggesting that calpain plays a modulatory role in T cell activation and production of Th1/Th2 type cytokines during the relapsing and remitting phase of some diseases [37, 49]. Moreover, it has been shown that calpain inhibitors can reduce TNF-α mRNA expression [50, 51] and proteasomal degradation of IκB and hence inhibit NFκB-driven transcription of pro-inflammatory cytokines and chemotactic factors . Meanwhile, inhibiting calpain by overexpressing a minimal domain of calpastatin could also coordinately suppress IL-1β and IL-6 activities [53, 54]. In line with these studies, we have shown here that inhibition of calpain by calpain inhibitor, MDL28170, reduced the levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and inflammatory transcription factor (NFκB) after TBI, but increased the levels of anti-inflammatory factors IL-10 and IL-4. The neuroprotective microenvironment attributed to the pretreatment with MDL28170, 30 min after TBI and before BMSC transplantation, may be of benefit to enhance the survivability of transplanted cells. Calpain inhibitors have been reported to inhibit both apoptosis and necrosis [55, 56], have neuroprotective effects in numerous rodent neurotrauma models, including TBI, spinal cord injury , and focal cerebral ischemia [45, 56, 57]. In fact, treatment with MDL28170 rescued transplanted BMSCs in the injured spinal cord by modulating ER stress-induced apoptosis . MDL28170 also enhanced the survival of transplanted Schwann Cells 7 days after transplantation into the contused spinal cord . Similarly, we demonstrated that MDL28170 pretreatment could reduce cell apoptosis and significantly enhanced the survivability of transplanted BMSCs after TBI compared with the BMSCs-only group. Therefore, these results support the use of calpain inhibitors as a promising new treatment for promoting the survival of transplanted cells.
The fact that a reduction in brain damage after TBI has been shown via BMSC transplantation alone [59, 60] corroborates with our data from this study. However, there is no significant decrease of lesion cavity in the MDL28170-only treatment group compared with the TBI group. This lack of effect on lesion volume has been seen with other calpain inhibitors, suggesting that pharmacological calpain inhibition alone though able to reduce axonal injury, may not in fact produce a measurable reduction in lesion volume [52, 61]. To the best of our knowledge, the combinatorial effects of MDL28170 and transplantation of BMSCs have not been investigated. Here, we showed that the pretreatment of MDL28170 followed by BMSC transplantation could achieve at least a 30% improvement in lesion volume compared with the BMSCs-only or MDL28170-only groups at 7 days after TBI. This may be due to the enhanced survival ratio of transplanted BMSCs and the neuroprotective effect exerted by MDL28170. Previous studies have also shown that MDL28170 was able to reduce motor neuron death and improve locomotor function . We demonstrated that the combination of MDL28170 and transplanted BMSCs saw a more distinct recovery of neurological function versus transplanted BMSCs alone, especially in the long-term study, which may be attributable to the anti-neurodegeneration and anti-inflammatory effects of the calpain inhibitor MDL28170. Taken together, our present work strongly suggests that the combination of calpain inhibitor pretreatment followed by cell transplantation produces more robust neuroprotective and functional recovery effects than either agent used alone and therefore warrants further study. For instance, to further elucidate the neuroprotective mechanism of the calpain inhibitor MDL28170, long-term experiments aiming to observe the number, localization, and differentiation status of transplanted cells in the lesioned brain are needed. Also, to study the mechanism of functional brain recovery more in-depth, we would suggest examining the regulation of neurotrophic factors, possible axonal regeneration and angiogenesis, and the potential formation of networks between endogenous neurons and transplanted stem cells differentiated neurons. Lastly, additional observations involving larger cohorts are required soon, with more definite conclusions regarding the safety of stem cell treatment to be made.
This study is the first to evaluate the use of MDL28170 combined with BMSC transplantation after TBI. Our data suggest that a single dose of MDL28170 in the acute phase of TBI improves the microenvironment by inhibiting inflammatory processes, which facilitated the survival of grafted BMSCs, leading to the reduction of lesion volume and the improvement of neurological function. Thus, we suggest a novel therapeutic strategy for TBI treatment by using a combination of MDL28170 and BMSCs. This promising new approach for promoting the survival of transplanted stem cells may be immensely beneficial for TBI patients relying on cell-based regenerative medicine.
The authors thank Dr. Kunlin Jin, University of North Texas Health Science Center, Texas, USA, for his invaluable support in experimental design and data analysis. The authors also thank Dr. Chun-Li Zhang, University of Texas Southwestern Medical Center, Texas, USA, for his enormous support in experimental design and manuscript editing.
This work was supported by the National Natural Science Foundation of China (No. 81771262), Zhejiang Health Science and Technology Project (2016RCA022), Zhejiang Key Research and Development Project (2017C03027) and American Heart Association Predoctoral Fellowship for Jiangnan Hu (19PRE34380114).
Availability of data and materials
All data generated or analyzed during this study are included in the published article.
JH, LC, XH, KW, and SD designed and performed the experiments, as well as analyzed and interpreted the data. JH, LC, WW, BW, CS, CR, and HN wrote and edited the manuscript. QZ, JY, and JH supervised the project and provided critical input. All authors gave feedback and agreed on the final version of the manuscript.
All animal experiments performed in accordance with the institutional guidelines for animal research, and approved by the Animal Care Committee of Wenzhou Medical University (China).
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- 12.Nichols JE, Niles JA, DeWitt D, Prough D, Parsley M, Vega S, Cantu A, Lee E, Cortiella J. Neurogenic and neuro-protective potential of a novel subpopulation of peripheral blood-derived CD133+ ABCG2+CXCR4+ mesenchymal stem cells: development of autologous cell-based therapeutics for traumatic brain injury. Stem Cell Res Ther. 2013;4:3.CrossRefGoogle Scholar
- 16.Skardelly M, Gaber K, Burdack S, Scheidt F, Hilbig H, Boltze J, Forschler A, Schwarz S, Schwarz J, Meixensberger J, Schuhmann MU. Long-term benefit of human fetal neuronal progenitor cell transplantation in a clinically adapted model after traumatic brain injury. J Neurotrauma. 2011;28:401–14.CrossRefGoogle Scholar
- 18.Lee JP, Jeyakumar M, Gonzalez R, Takahashi H, Lee PJ, Baek RC, Clark D, Rose H, Fu G, Clarke J, McKercher S, Meerloo J, Muller FJ, Park KI, Butters TD, Dwek RA, Schwartz P, Tong G, Wenger D, Lipton SA, Seyfried TN, Platt FM, Snyder EY. Stem cells act through multiple mechanisms to benefit mice with neurodegenerative metabolic disease. Nat Med. 2007;13:439–47.CrossRefGoogle Scholar
- 19.Redmond DE Jr, Bjugstad KB, Teng YD, Ourednik V, Ourednik J, Wakeman DR, Parsons XH, Gonzalez R, Blanchard BC, Kim SU, Gu Z, Lipton SA, Markakis EA, Roth RH, Elsworth JD, Sladek JR Jr, Sidman RL, Snyder EY. Behavioral improvement in a primate Parkinson’s model is associated with multiple homeostatic effects of human neural stem cells. Proc Natl Acad Sci U S A. 2007;104:12175–80.CrossRefGoogle Scholar
- 34.Kunz S, Niederberger E, Ehnert C, Coste O, Pfenninger A, Kruip J, Wendrich TM, Schmidtko A, Tegeder I, Geisslinger G. The calpain inhibitor MDL 28170 prevents inflammation-induced neurofilament light chain breakdown in the spinal cord and reduces thermal hyperalgesia. Pain. 2004;110:409–18.CrossRefGoogle Scholar
- 53.Iguchi-Hashimoto M, Usui T, Yoshifuji H, Shimizu M, Kobayashi S, Ito Y, Murakami K, Shiomi A, Yukawa N, Kawabata D, Nojima T, Ohmura K, Fujii T, Mimori T. Overexpression of a minimal domain of calpastatin suppresses IL-6 production and Th17 development via reduced NF-kappaB and increased STAT5 signals. PLoS One. 2011;6:e27020.CrossRefGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.