Many studies have observed the positive effects of minocycline as a neuroprotectant that can improve cognitive disorders in TBI rats. In this study, TBI rats that received minocycline therapy showed improved cognitive function through the NOR test, with significant improvements seen in the 50 mg/kg and 100 mg/kg dose groups (Fig. 2). This result is consistent with the findings of Kumar et al., [18] which support the role of minocycline in improving cognitive function with a dose of 50 mg/kg via the oral route. The oral route was chosen because it is easy to use in the long term, has a smaller risk of side effects such as irritation or infection when given repeatedly, and is easy to apply in humans.
This study observed the effect of minocycline specifically on astrocyte cells which are known to play a role in glial scar formation by creating an environment that is not ideal for CNS regeneration. The presence of astrocyte cells is indicated by GFAP which, if its intensity increases, will support the picture of glial scar formation. Figure 3B shows that minocycline treatment causes a significant decrease in GFAP intensity in the brain in the perilesion area in the 50 mg/kg and 100 mg/kg dose groups. The effect of minocycline administration for two weeks is to inhibit glial scar partially, not to prevent glial scar formation completely. This supports the goal of astrocyte-targeting therapy by maintaining the beneficial effects of astrocytes in the acute phase and correcting the deleterious effects in the chronic phase [2]. In the acute phase, the positive role of astrogliosis is still required to localize the injury process. In the chronic phase, the degree of astrogliosis is suppressed so that it does not reach a severe degree, thus it is hoped that the astroglial resolution process can still occur.
In this study, it was proven that the administration of minocycline significantly reduced the intensity of CNTF in TBI rats starting at doses of 25, 50, and 100 mg/kg (Fig. 4B). This shows that the effect of minocycline on CNTF is strong and confirmed with the smallest dose of minocycline, 25 mg/kg, a significant inhibitory effect of CNTF has been obtained. Previous research conducted by Tanaka et al., [16] on multiple sclerosis disease in the brain has also proven a significant decrease in CNTF mRNA levels in rats that received minocycline treatment for two weeks in the remyelination phase.
Figure 5B shows that the group of TBI rats that received minocycline treatment at a dose of 100 mg/kg experienced a significant decrease in TGF-β1 intensity. Previous research by Ataie-Kachoie et al. [15] on ovarian cancer also showed that minocycline treatment can inhibit TGF-β1. According to that study, TGF-β1 expression can be downregulated by administering minocycline, which targets NF-kB via suppressing TGF-β1-TAK1-IkB signaling.
In this study, there was a trend toward a decrease in the intensity of NF-κB p65 in the TBI rat group treated with minocycline. However, minocycline administration was not statistically significant in inhibiting the intensity of NF-κB p65 (Fig. 6B). The insignificant relationship between minocycline and inhibition of NF-κB p65 intensity does not follow the results of previous studies that proved the role of minocycline in inhibiting NF-κB pathway activation in ischemia disease [19], post-traumatic stress disorder [20], schizophrenia [21], Alzheimer's [22], spinal disease [23], and bone cancer pain [24]. The difference in the results of this study with previous studies may be due to differences in disease pathophysiology and different target organs.
Among the three signal pathways studied, minocycline significantly inhibits the formation of glial scar by inhibiting the CNTF and TGF-β1 pathways. In pathway analysis results, CNTF significantly influences GFAP positively by 63.8% (Fig. 7). Inhibition of CNTF by minocycline leads to a decrease in the canonical JAK-STAT3 pathway, where STAT3 is involved in a complex signaling cascade within the cell resulting in decreased expression of GFAP and astrocyte hypertrophy, thus inhibiting the formation of glial scar. Meanwhile, TGF-β1 significantly influences GFAP positively by 23.3% (Fig. 7). Inhibition of TGF-β1 will dampen the TGF-β/SMAD signaling pathway which will reduce the formation of glial scar and support intrinsic axonal growth and regeneration [25]. In this study, minocycline does not significantly inhibit NF-κB p65, but when interacting with CNTF and TGF-β1, NF-κB p65 plays a role in the mechanism of inhibiting glial scar by 24.5% (Fig. 7). Inhibition of minocycline in two out of five signal pathways of glial scar formation mechanisms can explain the mechanism of partial inhibition of glial scar formation rather than total inhibition.
Inhibition of glial scar is believed to be an effective strategy in cognitive function recovery. In this study, the significant impact of inhibiting glial scar on cognitive function improvement is found to be 50.4% (Table 1). Although glial scar inhibition is quite dominant, there are various other intrinsic and extrinsic factors contributing to cognitive function recovery. Intrinsic factors of nerve cells involved in axon regeneration include the expression of phosphatase and tensin homolog (PTEN) and suppressor of cytokine signaling 3 (SOCS3), while extrinsic factors include oligodendrocytes, meningeal cells, and the extracellular matrix (ECM). These inhibitory molecular factors bind to receptors expressed on elongating neurites, indicating a decrease in growth cone and axon regeneration cessation [26].
To follow up on the findings of this research, several further observations are needed. Among these are longer observation periods with extended minocycline administration via oral dosing variations. Confirmation of clinical improvement in brain-injured rats will be conducted more comprehensively through the observation of other neurobehavioral functions such as affective testing and motor testing. We also suggest further research on Chondroitin Sulfate Proteoglycans (CSPG) produced by glial scar, which chemically inhibits axon regeneration. To enhance cognitive improvement, inhibition of glial scar can be combined with therapy aimed at improving intrinsic CNS function by inducing pro-regenerative gene expression and grafting neural progenitor cells (NPC).