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56 (); 50-56
doi:
10.1016/j.jor.2024.05.020

Establishment of intervertebral disc degeneration models; A review of the currently used models

,
a Cell Therapy and Experimental Surgery of Musculoskeletal System LR18SP1 Lab, Faculty of Medicine, Sfax, Tunisia
b Department of Orthopedics and Traumatology, CHU Habib Bourguiba, Sfax, Tunisia

⁎Corresponding author: Najah Elmounedi. najah.mounedi12@gmail.com

Received: , Accepted: ,
Professor P K Surendran Memorial Education Foundation
Disclaimer:
This article was originally published by Reed Elsevier India Pvt. Ltd. and was migrated to Scientific Scholar after the change of Publisher.

Abstract

Abstract

One of the frequent causes of low back pain is intervertebral disc degeneration (IDD), which is followed by discogenic pain. Some significant risk factors that have been linked to the onset and progression of IDD include age, mechanical imbalance, changes in nutrition and inflammation. According to recent studies, five types of animal models are established for producing IDD: the spontaneous models, the puncture models, the biomechanical models, the chemical models and the hybrid models. These models are crucial in studying and understanding IDD's natural history and identifying potential treatment targets for IDD. In our study, we'll talk about the technical aspects of these models, the time between model establishment and the apparition of observable degradation, and their potential in various research. Each animal model should be compared to the human natural IDD pathogenesis to guide future research efforts in this area. By improving knowledge and appropriate application of various animal models, we seek to raise awareness of this illness and further translational research.

Keywords

Intervertebral disc degeneration
Animal model
Research methods
Surgery method
1

1 Introduction

Low back pain (LBP) linked to degeneration of the intervertebral disc (IVD) is a widespread public health problem affecting a large proportion of our aging population.1 They represent the most frequent causes of LBP and acute and/or chronic radiculalgia. They constitute by their frequency and their socioeconomic burden a public health problem also having significant economic repercussions due to the numerous days of work stoppage and the resulting daily allowances. LBP affects nearly 600 million people worldwide.2 This lumbar pain is explained in 40 % of cases by lumbar intervertebral disc degeneration (IDD).3 These important and worrying figures encourage the early detection of pathological disc degeneration. At this stage of the disease, it is possible to set up some successful clinical protocols to slow down or even stop the progression of IDD. The IVD is composed of 3 histologically different components: nucleus pulposus (NP), annulus fibrosus (AF), and cartilage endplate (CEP). Various factors contribute to IDD, including cellular senescence, alterations in genetic, biomechanics, cellular activity, and, in nutrition via the limited vascular supply … etc.4

Along with human research, animal research has contributed appreciably to understanding different physiological aspects and disease processes affecting human beings. The use of animals in experimentation is based on scientific, ethical, and legal justifications underpin. Maintaining these 3 principles in balance will contribute to the success of the research work. Research models must be standardized and apt to imitate human clinical conditions.5

Numerous techniques have been utilized to reproduce the features of IDD, to enhance comprehension of its processes, and for the pre-clinical assessment of new therapeutic approaches, which can be designed to relieve pain and reinstate function and biomechanics of IVD. Using in vitro models can be preferable as compared to using in vivo models because of (i) shorter experimentation times, (ii) more cost-effectiveness (iii) more control over experimental circumstances, and (iv) ethical concerns.6

In this review, we attempted to include the benefits and drawbacks of these models, the time required to build them, and other essential technical information. Lastly, we expect that this review will support the creation of novel therapeutic approaches and aid in the right selection of IDD models.

2

2 Animal models of IDD

Due to the complexity of IVD and the resulting degenerative changes with age, it has proven difficult to faithfully reproduce the complex physiological and pathophysiological mechanisms of IDD observed in humans. Animal models are important preclinical tools for biomedical research. Ideally, the most appropriate animal model for a specific disease involves artificially developing a condition that replicates the human condition. Due to the complicated etiology and pathogenesis of IDD, the development of a suitable animal model is difficult because it must i) share similar characteristics to IDD in humans; and ii) also must be reproducible, reliable, easy to maintain, and inexpensive.

The use of animal models is essential for the understanding of IDD as well as the pre-clinical evaluation of new therapeutic strategies. Two categories of animal models are described including spontaneous models and induced models.7 Spontaneous models include animals that spontaneously develop IDD in direct relation to aging, genetic alteration, or specific reproductive conditions. Induced models are subdivided into models based on mechanical or structural damage (direct alterations of the IVD using injury, inflammation, or chemical damage). It is essential to choose an induction model adapted to the disease under study.

2.1

2.1 Spontaneous models of IDD

The spontaneous model of IDD is theoretically closer to human disc physiopathology. The results of animal studies appear interesting. The sand rat (Psammomys obesus), subjected to a special diet, presents spontaneous degeneration of its IVDs. It constitutes the leader of these models.8 The other models are obtained with transgenic animals (rat or mouse) having a mutation in a coding gene, for example for type II collagen9 IX10 or carrying the allele HLA-B27 classically found in ankylosing spondylitis.11

Furthermore, a cellular matrix protein named SPARC (Secreted protein acidic and rich in cysteine) participates in the deposition of collagen, interactions between cells and extracellular matrix (ECM), and remodeling of ECM.12 IVD cells from aged IDD participants showed lower levels of SPARC expression.13 SPARC-null mice demonstrated defective connective tissue and accelerated IDD.14 Moreover, SPARC-null mice showed signs of behavioral symptoms indicating chronic lower back and radicular pain 15. The development of behavioral symptoms of axial and radiating LBP and diminished physical function increased with aging in SPARC-null mice.16 Furthermore, the small size of the IVDs of these animals constitutes a real limitation which makes it difficult to obtain sufficient quantities of tissue to allow biological analyses, most of the time requiring the grouping of material from several IVDs. This difficulty can be major with regard to complex surgical procedures.

2.2

2.2 Biomechanical models

Biomechanical models induce IDD by increasing the stresses experienced by a target IVD, either by an external stressing device,17 or by increasing its joint range of motion.18 These models induce a process relatively close to the pathophysiology of IDD but at the cost of painful handling for the animal. They are nevertheless interesting for obtaining reliable information on the IDD process. Mechanical animal models cause IDD by altering the typical biomechanical state.

2.2.1

2.2.1 Model of spinal instability

The cervical, thoracic, and lumbar spines were exposed, and the posterior paravertebral muscles were detached Subsequently, the connected supraspinous and interspinous ligaments as well as the spinous processes were removed. The model is known as the lumbar spine instability (LSI) model. It was frequently utilized to build the IDD model in the lumbar spine. Xue et al. used this model to investigate the function of skeletal interoception in establishing cartilage endplate degeneration and spinal-associated pain 19. A recent study showed that the LSI model was conducted for spinal hypersensitivity in dorsal root ganglion, which explains the pain that IDD promotes.20

In building the model of coccygeal spine instability, Bian et al. eliminated the NP after stepping over the entire depth of the Co7/8 AF.21 Four weeks after surgery, a histological examination of the adjacent Co8/9 IVD verified the IDD model's successful establishment.

2.2.2

2.2.2 The model of a tail looping

Tail-looping model is a new technique for constructing a model of IDD by establishing force imbalance within the IVD. Sakai et al. used 0.8-mm stainless steel wire to fixate the Co5 and Co13 vertebrae after looping the mice's tails. They removed the additional vertebrae. Aspirating the NP of the Co7/8 and Co8/9 IVDs produced more severe degeneration.22 The Co10/11 and Co11/12 IVDs were selected as slightly degenerate IVDs in this model, while the Co2/3 and Co3/4 IVDs were chosen as control IVDs. Researchers found that deterioration started as early as 8 weeks after looping.22 Another study joined the Co2 and Co9 vertebrae together using the tail-looping method to induce the IDD model.23

2.2.3

2.2.3 Axial-compressing external fixation devices

The IVDs may narrow and stiffer when subjected to compression and angulation.24 MacLean et al. attached external rings to adjacent IVD levels by percutaneously transfixing the vertebral bodies with 0.5 mm pins.25 This procedure was modified by Stokes et al. by adding rings at a 15-degree angle or parallel to one another.26 According to their findings, more disc space was lost when there was a 15-degree angulation combined with compression. Recently, Kirschner wire was inserted into the Co8 and Co10 vertebral bodies by Ji et al. to establish a unique device. The tail was then bent 40°, and the Co8/9 and Co9/10 IVDs were subjected to forces of 1.8 N and 4.5 N applied via springs.27 Two weeks post-surgery, Pfirrmann grades, and histological investigations demonstrated the existence of IDD. There is a positive correlation between the force applied and the degree of deterioration.27

Miyagi et al. induced IDD by using both the compression and the needle puncture models and evaluated pro-inflammatory factors that were increased. Furthermore, the increase in positive labeling of calcitonin gene-related peptide (CGRP) neurons, suggests a probable mechanism IDD contributes to LBP.28

2.2.4

2.2.4 Vibration model

Clinical findings proved that employees who are subjected to occupational vibration have an increased risk of developing IDD.29 Research has connected vibration exposure to decreased ECM production and increased matrix metalloproteinase release,30 indicating that vibration may be a risk factor for the progression of IDD. In a research conducted by McCann et al., mice were given a clinically utilized vibration frequency (45 Hz, peak acceleration of 0.3 g for 30 min/day and 5 days/week) for one month.31 After analysis, the morphologic grade, which was particularly marked by AF degeneration, verified the occurrence of the IDD onset. Moreover, they discovered that the mice's IDD was not reversed by 4-week WBV followed by a 4-week cessation, indicating that the damage is irreversible.32 Another study found that mice exposed to 8 Hz and 15 Hz WBV caused persistent cervical and lumbar discomfort,33 suggesting that the WBV model could be helpful in studies about pain 34.

2.2.5

2.2.5 Bipedal animal model

This model was created by amputating the forelimbs of rodents. A forced bipedal stance following forelimb amputation imitates a human's bipedal movement.35 A group of researchers carried out amputation surgery on male rats that were one month old and the rats were housed in specially designed cages that forced the rats to stand erect,36 five months post-amputation surgery, a decrease in cervical disc height (DH) was detected, seven months later, the DH loss was more severe. Additionally noted was the reduction in aggrecan and collagen type II expression.36 Likewise, Liu et al. found that ligustrazine attenuates cartilage endplate hypertrophy, a hallmark of IDD, during a nine-month observation period through the use of the bipedal rat model.37 Although this model is similar to IDD in humans, this model can take up to 6 months to achieve detectable IDD. An additional concern that prevents the application of this model is animal welfare, due to trauma caused by surgical amputation. The latter can also be the origin of the modification of the eating habits of animals.35

2.2.6

2.2.6 Ovariectomy

Deng et al. proved that lumbar IDD in female rats occurs six months following ovariectomy.38 Due to the long duration of the experiment, it is confirmed that disc needle puncture can accelerate the onset of IDD. In our study, we established an animal model of IDD via the combination of estrogen deprivation and IVD puncture.39

2.3

2.3 Chemical models

These models use chemo-nucleolytic substances that induce the destruction of collagen, proteoglycans (PG), aggregates and glycosaminoglycan (GAG) of the ECM such as trypsin 40, papain41–43, chymopapain44, monosodium iodoacetate (MIA),45 chondroitinase ABC,46 5-bromodeoxyuridine47 or inflammatory cytokines including TNF-alpha48 in the discs. The gravity of the resulting tissue deterioration depends on the substance concentration and exposure duration.

Exposure of bovine IVDs to papain 49,50 and porcine discs to chymopapain51, under unloaded conditions, results in cavities in the NP. While cracks in the NP have been detected in samples exposed to physiological loading during digestion through the use of papain or trypsin, cavities have not.52,53

Additionally, injections of papain and trypsin lessen the compressive (≃40 %) and rotational (>50 %) stiffness of the disc.52,53 Collagenase and elastase have also been utilized in biochemical models of IDD.54,55 Collagenase degrades collagen fibers, lowering the collagen/GAG content ratio in the IVD56 and resulting in tissue compaction, which raises compressive stiffness by around 10 %. Furthermore, samples treated with collagenase showed a considerable decrease in DH (≼40 %) when compared to the control IVD..54 Elastin degradation due to elastase injection reduces traction (25 %) and torsional stiffness (65 %) and DH (≃30 %).54,55 GAG losses were also detected in elastase-treated samples, which increased the amount of free amine.54

Far from the “natural” process of IDD, it seems inappropriate to use this type of model for pathophysiological studies. Indeed, substances that induce IDD risk causing interactions with cells and constituents of the ECM. However, studies aimed at evaluating certain therapies seem possible, always taking into account possible interactions with the inducing substance.

2.4

2.4 Disc puncture method in the IDD animal model

The needle puncture model is a structural disruption established via the puncture of the IVD from the anterior or posterior direction. This technique is frequently utilized for inducing IDD in animals such as mice,57 rats,58,59 rabbits,60 dogs,61 pigs,62 sheep63 bovine,64 and rhesus monkeys.65 This model is very simple and easy to establish. It only requires a puncture and rotation of the needle in the AF without disrupting the NP, so this method only requires light sedation of the animal.66 The selected disc can be detected by palpation and confirmed by X-ray monitoring. The depth of penetration can be determined by marking the used needle. The needles are habitually inserted into the IVD and left there for 30–60 s.66,67 In several studies, the needle was rotated for 180–360° after being placed in the IVD.68

In the puncture model, the effect of needle size, number of rotations and puncture depth are important factors influencing the onset and progression of IDD. It has also been noted that needle rotation results in faster IDD.69,70 Our experiments demonstrated that the 21G gauge needle makes it possible to establish a rapid IDD model (The signs appear one week after the puncture) one week post punction.71 This process is irreversible and continually progresses to advanced stages. This animal model has the benefits of minimal cost, a short experimental cycles, a high survival rates, excellent repeatability, and short timespan needed to produce the intended degenerative effect.72 Our study analyzed a degenerative cascade gradually produced through the use of various needle gauges and revealed stepwise changes until severe IDD was reached.71 A previous review article concluded that the ratio of needle diameter to perforated DH was found to be important for inducing considerable degeneration in discs, and it should be greater than 0.4.73 As greater trauma could result in more severe IDD, a puncture injury induced by 18G (ratio 0.97), 21G (ratio 0.63), or 23G (ratio 0.49) needles would result in severe and rapid IDD of the coccygeal IVDs of the rat (height 1.3 mm). Larger needle sizes (14G, 16G, 18G, or 19G) can intensively damage discs, producing rapid and severe IDD that is not compatible with progressive degeneration processes.74 Furthermore, 25G (ratio 0.39) is sufficient to produce significant but less severe IDD.71

AF lesions can be induced by an annular laceration model using a scalpel, a drill,75,76 or a more superficial injury using a needle stick.77–79 The size of the induced defect is decisive for the speed of appearance of the IDD; the larger the lesion, the faster the IDD. Thus the number of rotations of the needle can influence the grade of the IDD and its speed of installation. Perforation of the CEP has also been utilized to initiate IDD.118

The physiological and anatomical characteristics of the selected animal must be as close as possible to humans. The caudal IVD of Sprague Dawley rats is beneficial to use due to their similarity to the biochemical components of human IVDs.72,80,81 It was suggested to use the rat caudal IVD as a platform for the puncture model that produced IDD.70,72,82 Puncture can induce continued severe degeneration within an appropriate time frame while imitating the human degenerative cascade.72

It is currently accepted that puncture models produce lesions with high reproducibility whether inter-individually within the same study or between distinct studies using the same animal species. Additionally, the rat tails continued to function normally and the puncture site healed well without developing an infection. Moreover, this surgery requires a simple operation and offers practical research methodologies. This animal model created through percutaneous needle puncture method is expected to mimic the human IDD process.

Structural lesions can be made at the level of AF and/or NP. AF lesions can be obtained with a scalpel blade77 or by puncturing the AF with a needle.83 This model significantly lessens damage to lab animals while mimicking the process of progressive IDD. Even more important, this model proved that AF damage alone could lead to IDD.

In the case of NP lesions, aspiration of part of the NP is then performed.79 The induction of this type of lesion requires an invasive approach and leads to a degenerative process far removed from the pathophysiology of spontaneous degeneration. The study of physiopathology by this type of model does not seem very suitable. They nevertheless find, in part, an interest for studies of therapeutic efficacy to which we will return later.

The coccygeal disc puncture model is very widespread. Several recent studies use this model to evaluate the effect of new therapeutics.84–88

2.5

2.5 Hybrid techniques of IDD

IDD models classified as hybrids combine both biological and mechanical initiators. If the injection was paired with extra initiators, including mechanical loading or physical disruption of the IVD, it was considered a hybrid.

The IVD components (AF and NP) have been punctured 100 times to induce depressurization and annular rupture. Collagenase or MMP-3 injections have also been utilized to break down collagen and PGs.89,90 It was demonstrated that there was a decrease in DH that was almost 25 % larger in both the 0.5 % and 1.0 % collagenase groups compared to the control group after 30 k cycles. In contrast, the discs injected with PBS or MMP-3 caused an increase in DH.90

Differences in animal and human anatomy, disc size, cell type, and loading further complicate the search for an ideal animal model. Notochord cells disappear with maturity at age 10 in humans, representing an important milestone in IDD. Unlike the human disc, notochord cells are conserved in the majority of animal species,100 which manifests itself as a difference between human and animal models.

3

3 Experimental endpoints of IDD

3.1

3.1 Diagnostic imaging

The majority of studies utilized standard techniques to analyse the inhibition of IDD progression of the regenerative process such as radiography (X-ray), MRI, histopathology and immunohistochemistry (IHC) (Fig. 1).

Frequently employed diagnostic techniques for IDD diagnosis and treatment response assessment in several in vivo studies. IHC: immunohistochemistry; IVD: intervertebral disc; PCR: polymerase chain reaction; WB: Western blot; MRI: magnetic resonance imaging.
Fig. 1 Frequently employed diagnostic techniques for IDD diagnosis and treatment response assessment in several in vivo studies. IHC: immunohistochemistry; IVD: intervertebral disc; PCR: polymerase chain reaction; WB: Western blot; MRI: magnetic resonance imaging.

Magnetic resonance imaging (MRI) is considered the gold standard imaging method for the assessment of the spinal cord, IVD, paraspinal soft tissue structures, and other neurological structures.101 On T2-weighted MRI images of the spine, the healthy IVD shows high signal intensity in the NP (rich in water and PG), whereas the fibrocartilaginous AF shows low signal intensity.102 T2 hyperintensity in the NP is usually reduced in IDD, and the tissues surrounding each IVD (end plate sclerosis, vertebral osteophytes, and disc herniation) are altered.103 To further define IDD, MRI sequence analysis can yield quantitative (NP size, water content, etc.) and qualitative (IVD shape, EP alterations, etc.) data.

The most popular MRI grading system for the semiquantitative evaluation of the human lumbar IDD state was proposed by Pfirrmann et al. The grading system is based on the distinctive disc structural alterations (T2-weighted signal intensity, IVD structure, capacity to distinguish between NP and AF, and DH).104 Preclinical animal models of IDD use MRI more frequently due to researchers having easier access to this modality. Glaeser et al. have reported high-quality micro-MRI pictures of rat spines taken with the Bruker Biospec 9.4 T MRI Scanner.105 However, the majority of research has used significantly lower field strength imaging (1.0, 1.5, and 3.0 T) and lower-quality imaging.106

MRI, by its ability to provide information on both the morphology and physiology of soft tissues, is suitable for quantitatively and non-invasively accessing early and late changes due to IDD. Changes in the structure of the IVD are visualized in a clinical routine on images with T1 and T2 contrast. These two contrasts provide access to morphological information of the IVD such as the DH and the areas of the IVD and the NP. Changes in signal intensity on these images help identify degenerative discs. T1 contrast is used to identify anatomical abnormalities of the IVD, which occur in severe stages of IDD.107 The reduction in signal intensity on T2-weighted images is always observed with worsening signs of IDD. This decrease in T2-weighted signal is associated with the loss of water content of the IVD.77 Because of the results' capacity to produce quantitative comparisons between study cohorts, the use of these quantitative methodologies is becoming more and more common in IDD research. This modality's application in preclinical animal research raises crucial questions for future studies that will help fully characterize degeneration.

In the studies that were reviewed, radiography (X-ray) was the second most commonly utilized imaging modality. This is probably because it is more affordable, more accessible, and requires the least amount of technical expertise to execute and review. Although IVDs cannot be seen on radiographs. A DH index percentage (%DHI) has been described as an effective depiction of disc size for objective examination, with the DHI being assessed in preclinical animal models both pre and post-IDD-induction of IDD at specific intervals. As radiographs are non-destructive, this allows serial images to be easily taken, which might be helpful for comprehending how height decrease occurs over time. X-rays are restricted in the detail of data that can be extrapolated but remain a useful tool because of the ease with which films may be obtained and the possibility of obtaining serial films on living animals. Radiography is very useful as a non-invasive method to obtain images of IVD throughout the course of the experiment.

Other modalities, such micro-CT, provide better picture quality due to technological developments, making them more useful for diagnosing and identifying minute changes in discs and the surrounding supporting structures, like the facet joints and vertebral body.

Every imaging modality has benefits and drawback. X-ray is non-invasive, effective, and can easily be conducted at various stages of the IDD animal model to monitor progress. In contrast, despite being somewhat more expensive, micro-CT and MRI offer better quality images and are hence more sensitive in detecting degenerative changes related to IDD. Micro-CT allows for even greater resolution scanning of bone structure, which is necessary for investigation of small rodent spinal units.108 In order to provide further insight into the osseous and mineralized degenerative changes linked to IDD, micro-CT may be used to more thoroughly show the osseous anatomy, including evaluation of the internal osseous lamellar structure and vertebral endplates.

3.2

3.2 Histology

Various staining techniques can be used to reveal the disc's histological features, and established classification schemes can be used to give a grade to degenerative characteristics. The histological grading scale developed by Masuda et al. was widely used.69 This grading scale considers the following characteristics: the degree of AF fiber disruption, interruption of the AF-NP boundary, reduction in NP cellularity, and NP matrix condensation. Although it was frequently employed, this was not the only classification system used. Complete IVD histological grading methods for mice,109 rats,110 rabbits,111 and big animal models101 have been recently proposed.

For the rat, the histological scale proposed by Lai et al. is composed of 5 categories that were utilized to evaluate the cellularity of NP and AF, the morphology of NP of the AF and endplate, also the border between the two structures (NP and AF). Each category is noted from, « 0 », corresponding to a healthy disc, to « 2 », corresponding to a completely degenerated IVD. Therefore, the score varies from 0, for a normal disc, to 16 for a severely degenerated IVD.110

The grading methods were created following a review of the literature, a survey of clinicians and researchers with expertise in spinal research, and a validation study including both beginner and expert disc histology graders.112 By using these proposed grading systems, the authors hope to lower variability and increase the objectivity of histological analysis comparisons between preclinical animal models and studies. For histological investigation, hematoxylin and eosin (H&E) was the most widely used stain. It was frequently employed in conjunction with either Alcian-blue or Safranin-O/Fast green. The cytoplasmic and nuclear components of the disc and glycosaminoglycans can be identified using H&E and Alcian blue/Safranin-O, respectively, as baseline stains. Nevertheless, because of the intricate design of the disc compartments, triple-dye techniques, including Masson's trichrome, have recently been developed to boost histology resolution.113 Picrosirius red has also been included to detect the orientation of collagen fibers. In ABPR (alcian blue-picrosirius red) combination dye techniques using hematoxylin or Safranin-O for distinguishing staining of collagen (red), PGs (blue), and nuclear components.114,115

3.3

3.3 Gross morphology

Macroscopic assessment of IVDs is another effective approach to grading IDD. A direct visualization or high-resolution picture can be used to evaluate the IVD's primary structures. The most frequently used and cited macroscopic grading score in the examined literature was proposed by Thompson et al..116 This type of evaluation offers the advantages of being simple to carry out, inexpensive, and therapeutically relevant, since this kind of assessment was initially intended to evaluate human IVDs. It's important to remember that visual examination is intrinsically subjective, thus utilizing two separate assessors will reduce evaluation variability.101 Although macroscopic evaluation is not suitable for identifying minute changes, it is a straightforward method that researchers can use to get more comparison data from their study.

3.4

3.4 Biochemical analysis and gene or protein expression

Biochemical assessment can be used to characterize the process of IDD, identify specific metabolite products or changes in protein expression, and suggest potential targets for prevention or treatment. The hydroxyproline and dimethyl methylene blue (DMMB) assays, which measure the amount of collagen and GAG in the sample, were two prevalent quantitative biochemical assays used in the investigated literature. These tests are useful for emphasizing content levels, but they don't tell us anything about the caliber of the matrix content. As far as the authors are aware, there are no reference levels available for particular gene expression in the disc of certain animal species. Some of the difficulties associated with gene expression profiling in cartilaginous IVDs we noted: low cellularity and an ECM rich in GAGs that can impede real-time polymerase chain reaction (PCR).101 Utilizing experimental methods like Western Blot technique, PCR, enzyme-linked immunosorbent assays, and immunohistochemistry can provide a much more in-depth examination of IDD at the cellular level because gene expression levels can differ from protein levels.

When examining IDD, a variety of experimental endpoints can be used. Whether it is done macroscopically, microscopically using histology or other alternative staining techniques like immunofluorescence, biochemically, or using advanced imaging methods as previously stated, each technique evaluates a different element of IDD. Other endpoints that can be used, though less frequently, are methods like cell culture and biomechanical testing, which provide a variety of quantitative insights into different aspects of IDD. Future studies will examine the adequacy of experimental techniques for reliably assessing IDD to help standardize trials, boost translational application to human IVD health and disease, and achieve relatability of data.

3.5

3.5 In silico study “molecular docking”

Molecular docking is an in silico approach aimed at predicting and simulating the most favorable position of a ligand within the protein (target).117

4

4 Conclusion

To sum up, animal models are essential for comprehending, describing, and treating IDD. Nevertheless, there is still disagreement over which model most closely resembles IDD, even with the strategies described in this analysis. More critically, there is still some discrepancy between the clinical symptoms and model-induced IDD. More research is required to test the fidelity of these models and ultimately aid in the creation of novel therapeutic approaches.

Ethical statement

Not applicable.

Funding

This study received no external funding.

GuardianPatients consent

Not applicable.

CRediT authorship contribution statement

Najah Elmounedi: Writing – original draft, Software, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Hassib Keskes: Writing – review & editing, Supervision.

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