Spinal cord injury (SCI) is a complex condition that profoundly affects various physiological systems, leading to severe impairments in the sensory, motor, and autonomic functions.^1,2,3 SCI disrupts communication between the brain and body and affects the autonomic nervous system, leading to complications in cardiovascular, respiratory, urinary, gastrointestinal, and sexual functions.^2,4 Furthermore, SCI can lead to contradictory symptoms. For instance, although it generally causes motor paralysis and sensory loss, it can also result in spasticity, a condition characterized by increased muscle tone and exaggerated reflexes.⁵

The injury process begins with the primary mechanisms of trauma and involves the mechanical disruption of neural tissues, axons, and blood vessels.⁶ This initial insult leads to immediate necrotic damage at the injury site, often accompanied by hemorrhage and edema. However, the extent of the damage is not solely defined by this primary trauma; it is significantly amplified by secondary mechanisms that unfold over the hours, days, and weeks following injury. This secondary injury cascade involves complex molecular and cellular events, including ischemia (restricted blood flow), inflammation, oxidative stress, ionic dysregulation, and excitotoxicity, caused by excessive glutamate release. Together, these processes contribute to widespread neural cell death, demyelination, and scarring, ultimately leading to functional deficits in patients with SCI (Figure 1).^6,7,8

Secondary injury mechanisms are interconnected and self-perpetuating, resulting in progressive damage.⁹ Inflammation exhibits a dual nature: while crucial for removing cellular debris, it releases pro-inflammatory cytokines and reactive oxygen species, exacerbating tissue damage.^7,8,9,10,11 Oxidative stress, caused by an imbalance between free radicals and antioxidants, compromises cellular components including lipids, proteins, and DNA.^12,13 Mitochondrial dysfunction impairs energy production and initiates apoptotic pathways.^14,15 The accumulation of calcium ions within neurons disrupts cellular functions and contributes to neuronal death. Despite these deleterious mechanisms, certain factors may have regenerative potential. Specific inflammatory signals can stimulate the activation of endogenous neural stem and progenitor cells,^16,17,18,19 which migrate to the injury site and differentiate into neurons and glial cells. However, their regenerative potential remains insufficient without therapeutic intervention.^7,16,20 In SCI models, endogenous NSPCs are activated in the neural tissue adjacent to the injury and migrate to the epicenter.²¹ Although NSPCs can differentiate into functional neurons in vitro, their in vivo neuronal differentiation is limited. Nakagomi et al. showed that in a stroke model, nestin-positive cells from the stroke-affected cortex migrated to the peri-infarct area and differentiated into glial cells; however, neuronal differentiation was not detected in vivo,²² highlighting the influence of the microenvironment on NSPC fate.

Current research emphasizes targeting multiple pathways within the secondary injury cascade as single-mechanism approaches may not yield substantial clinical benefits.^23,24,25,26 Therapeutic interventions targeting inflammation, oxidative stress, and excitotoxicity show potential in preclinical studies,^23,24 including anti-inflammatory drugs, antioxidants, calcium channel blockers, and neuroprotective agents. Regenerative therapies, including stem cell transplantation, gene therapy, and bioengineered scaffolds, aim to enhance neural tissue repair.^23,24,26,27

The complexity of SCI pathophysiology requires diverse laboratory models to investigate its mechanisms and evaluate interventions.^28,29 Animal models are categorized by injury conditions like contusion, compression, dislocation, transection, or chemical damage.^28,30,31 Rodents are widely used for accessibility, cost-effectiveness, and genetic manipulability,³² while larger animals, including primates, porcine models, and canines, better approximate human SCI characteristics.^29,33,34 Each model provides insights into different aspects of SCI pathology.

Animal models have helped to identify therapeutic targets and evaluate experimental interventions, including pharmacological agents, stem cells, and neuroprosthetics. These preclinical studies have established the foundation for clinical trials, although translation to humans remains challenging owing to biological differences. Ongoing advances in understanding SCI pathophysiology and developing innovative therapies offer promise for improving outcomes in affected individuals.

A systematic literature review was conducted to construct a comprehensive overview of experimental SCI models. This review aimed to collect, synthesize, and critically evaluate the existing in vivo, ex vivo, and in vitro SCI models, emphasizing their translational relevance, experimental utility, and limitations.

In vivo models using live animals are essential to replicate SCI’s mechanical and physiological aspects of SCI. Rodent models are invaluable for understanding mechanisms and evaluating therapeutic interventions.²⁸ Although rodents are most commonly used, larger animals, such as pigs and monkeys, are employed for translational research.^33,35

The choice of species influences translational potential. Rodents are preferred for cost-effectiveness and genetic modifiability; however, their spinal cord size and immune responses differ from those of humans. Larger animals offer better anatomical similarities to humans despite ethical and cost challenges. Selecting appropriate species and models is crucial for research advancement. SCI models provide tools to explore injury and repair mechanisms through transection, hemisection, chemical lesions, and ischemic models. Although promising results exist, the complexity of SCI requires tailored approaches. Integrating insights from various models can help advance therapeutic interventions (Table 1).^{31,36,37,38,39,40}

Ex vivo SCI models provide platforms for investigating spinal cord dynamics in controlled environments by maintaining extracted tissue in culture systems. These models address the ethical challenges of in vivo studies using localized injury response analysis. Weightman et al. demonstrated organotypic slice arrays for replicating cellular responses to injury, including gliosis and nerve fiber outgrowth, thereby reducing invasive procedures.⁷¹ Integrating aligned polylactic acid nanofiber meshes enabled the assessment of the regenerative potential of nanomaterials. Yan et al. developed a micropatterned conductive nanofiber mesh with PCL and NGF, facilitating nerve stem cell differentiation while suppressing astrocyte formation.⁷² The aligned nanofibers guided neurite outgrowth, mimicking in vivo patterns, and demonstrating translational relevance.

Explant models reveal neural regeneration processes including cell survival, differentiation, migration, and axonal regrowth. Dorsal root ganglia explants have shown that oxidized galectin-1 promotes axonal regeneration and Schwann cell migration.⁷³ A three-dimensional DRG model has demonstrated the role of the urokinase system in neural cell migration and axonal branching.⁷⁴ Studies on Schwann cell migration and axonal regrowth have shown conflicting results regarding the process sequence after nerve transection.⁷⁵ Explant models help study neuronal-Schwann cell interactions and nerve regeneration factors.^76,77

Human ex vivo spinal cord slice cultures provide a model to study neuronal populations and inflammatory responses. These cultures maintain neuronal subpopulations and preserve the tissue cytoarchitecture.^78,79 Ventral horn motoneurons decline after 3 days, while calbindin-positive neurons persist longer.⁸⁰ IL-1β can activate endogenous neural progenitors of oligodendrocyte lineage.⁸¹ These models examine disease mechanisms such as neuromyelitis optica through immune cell-cytokine interactions.⁸²

Ex vivo models, including organotypic slice cultures, offer key advantages. Controlling variables, such as temperature and nutrients, enhances reproducibility and reduces confounding factors. These models are ideal for imaging and drug screening studies while reducing animal use.

However, ex vivo models cannot replicate systemic interactions such as immune responses and hormonal signaling, limiting extrapolation to living organisms. However, the limited survival time of tissue cultures impedes long-term studies. These limitations necessitate combining ex vivo and in vivo approaches to understand the pathophysiology of SCI.

In vitro SCI models use isolated cell cultures to investigate cellular and molecular mechanisms of injury and repair. These models provide controlled environments for understanding SCI processes and drug development. In vitro approaches include various systems with different applications and limitations.^83,84,85,86

The study of SCI requires diverse models to address its complex pathophysiology and to support the development of effective therapeutic strategies. In vivo, ex vivo, and in vitro models each provide unique advantages for exploring various aspects of SCI, including mechanisms of injury, repair processes, and therapeutic efficacy. In vivo models, particularly in rodents and larger animals, remain critical for replicating the physiological and mechanical characteristics of human SCI and understanding systemic factors such as inflammation, vascular responses, and functional recovery. Ex vivo systems, such as organotypic slice cultures and explant models, offer valuable tools for investigating localized cellular responses and regenerative processes within a controlled environment, while reducing reliance on live animals. In vitro models, including primary cell cultures, immortalized cell lines, and advanced systems such as organ-on-a-chip technologies, enable precise mechanistic studies and high-throughput screening for drug development.

Integrating emerging innovations such as iPSCs, bioengineered 3D cultures, and in silico simulations has transformed the landscape of SCI research. iPSC-derived platforms provide personalized approaches for disease modeling and therapeutic testing. In contrast, bioengineered systems replicate the structural and mechanical properties of the spinal cord and offer spatially relevant environments for regenerative studies. In silico models further complement experimental approaches by providing predictive insights that streamline experimental design and accelerate therapeutic development. These innovations address the limitations of traditional methods and improve the translational relevance of the preclinical findings.

However, each model has its inherent limitations. In vivo systems face inter-species differences and reproducibility challenges, whereas in vitro and ex vivo models lack systemic interactions, such as blood flow and immune responses, which are critical to SCI pathophysiology. A multifaceted approach that integrates complementary models is essential for overcoming these challenges. Combining the strengths of diverse methodologies will enable a comprehensive understanding of SCI mechanisms, facilitate the identification of therapeutic targets, and enhance the translation of promising interventions into the clinical setting.

In conclusion, continued advancement and refinement of SCI models and innovative technologies, such as bioengineered platforms, iPSCs, and computational simulations, are promising for improving the accuracy, relevance, and clinical applicability of preclinical research. By leveraging these tools, researchers can bridge the gap between experimental findings and clinical outcomes, ultimately paving the way for novel and effective therapeutic interventions to address the profound impact of SCIs.