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Traumatic damage to the spinal cord (SCI) frequently leads to irreversible neurological deficits, which may be related to apoptotic neurodegeneration in nerve tissue. The MLC901 treatment possesses neuroprotective and neuroregenerative activity. This study aimed to explore the regenerative potential of MLC901 and the molecular mechanisms promoting neurogenesis and functional recovery after SCI in rats. A calibrated forceps compression injury for 15 s was used to induce SCI in rats, followed by an examination of the impacts of MLC901 on functional recovery. The Basso, Beattie, and Bresnahan (BBB) scores were utilized to assess neuronal functional recovery; H&E and immunohistochemistry (IHC) staining were also used to observe pathological changes in the lesion area. Somatosensory Evoked Potentials (SEPs) were measured using the Nicolet® Viking Quest™ apparatus. Additionally, we employed the Western blot assay to identify PI3K/AKT/GSK-3β pathway-related proteins and to assess the levels of GAP-43 and GFAP through immunohistochemistry staining. The study findings revealed that MLC901 improved hind-limb motor function recovery, alleviating the pathological damage induced by SCI. Moreover, MLC901 significantly enhanced locomotor activity, SEPs waveform, latency, amplitude, and nerve conduction velocity. The treatment also promoted GAP-43 expression and reduced reactive astrocytes (GFAP). MLC901 treatment activated p-AKT reduced p-GSK-3β expression levels and showed a normalized ratio (fold changes) relative to β-tubulin. Specifically, p-AKT exhibited a 4-fold increase, while p-GSK-3β showed a 2-fold decrease in T rats compared to UT rats. In conclusion, these results suggest that the treatment mitigates pathological tissue damage and effectively improves neural functional recovery following SCI, primarily by alleviating apoptosis and promoting neurogenesis. The underlying molecular mechanism of this treatment mainly involves the activation of the PI3K/AKT/GSK-3β pathway.

Abstract Background Excitotoxicity-induced in vivo injury models are vital to reflect the pathophysiological features of acute spinal cord injury (SCI) in humans. The duration and concentration of chemical treatment controls the extent of neuronal cell damage. The extent of injury is explained in relation to locomotor and behavioural activity. Several SCI in vivo methods have been reported and studied extensively, particularly contusion, compression, and transection models. These models depict similar pathophysiology to that in humans but are extremely expensive (contusion) and require expertise (compression). Chemical excitotoxicity-induced SCI models are simple and easy while producing similar clinical manifestations. The kainic acid (KA) excitotoxicity model is a convenient, low-cost, and highly reproducible animal model of SCI in the laboratory. The basic impactor approximately cost between 10,000 and 20,000 USD, while the kainic acid only cost between 300 and 500 USD, which is quite cheap as compared to traditional SCI method. Methods In this study, 0.05 mM KA was administered at dose of 10 µL/100 g body weight, at a rate of 10 µL/min, to induce spinal injury by intra-spinal injection between the T12 and T13 thoracic vertebrae. In this protocol, detailed description of a dorsal laminectomy was explained to expose the spinal cord, following intra-spinal kainic acid administration at desired location. The dose, rate and technique to administer kainic acid were explained extensively to reflect a successful paraplegia and spinal cord injury in rats. The postoperative care and complication post injury of paraplegic laboratory animals were also explained, and necessary requirements to overcome these complications were also described to help researcher. Results This injury model produced impaired hind limb locomotor function with mild seizure. Hence this protocol will help researchers to induce spinal cord injury in laboratories at extremely low cost and also will help to determine the necessary supplies, methods for producing SCI in rats and treatments designed to mitigate post-injury impairment. Conclusions Kainic acid intra-spinal injection at the concentration of 0.05 mM, and rate 10 µL/min, is an effective method create spinal injury in rats, however more potent concentrations of kainic acid need to be studied in order to create severe spinal injuries.