Our current results demonstrate that SCI results in a marked, chronic upregulation of the expression of cell cycle-related proteins associated with reactive astrocytosis and microglial proliferation. Delayed systemic administration of CR8 limited chronic upregulation of cell cycle proteins and improved functional recovery up to 4 months post-SCI; this was associated with reduced astrogliosis and chronic inflammation that may contribute to the observed progressive tissue loss and glial scar formation.
SCI causes secondary biochemical changes that persist for months after SCI. The role of reactive astrocytes in the restorative stage after injury is complex, as they secrete numerous bioactive substances − including cytokines, antioxidants, recognition molecules and growth factors − that can be either neurotrophic or neurotoxic [43, 44]. GFAP expression and immunoreactivity were increased at 1 month and 4 months after SCI. Several cell cycle proteins were upregulated in GFAP+ reactive astrocytes concentrated in the boundary zone between spared tissue and the lesion; treatment with a specific CDK inhibitor after SCI reduced the sustained upregulation of cell cycle protein expression as well as GFAP immunoreactivity. Taken together, our data demonstrate chronic cell cycle activation in reactive astrocytes after SCI, which may contribute to the glial scar formation. Thus, the ability of cell cycle inhibitors to limit scar formation may facilitate endogenous restorative potential.
Recent studies demonstrated a secondary peak of inflammation as late as 2 months post-injury [45, 46]. We have shown that SCI in the rodent is followed by sustained upregulation of a cluster of proinflammatory genes for up to 6 months that may contribute to the continuation of damage in the injured cord [16, 17]. Although microglia have both neurotoxic and neuroprotective effects [20, 47, 48], considerable experimental data suggest that post-traumatic inflammation, including microglial activation, contributes to chronic cell damage and progressive tissue loss [30, 49, 50]. Indeed, activated microglia and release of associated inflammatory factors has been indicated as an important contributing factor for many acute and chronic neurodegenerative disorders [51, 52]. The present study confirms similar results evidenced by increased expression and immunoreactivity of the inflammatory markers, Iba-1, CD11b and a core component of the NADPH oxidase enzyme, p22PHOX; the increased cell cycle protein expression observed was co-expressed with these inflammatory markers in activated microglia as late as 4 months after SCI. In agreement with our previous findings [23, 24, 34], we detected reduction of inflammation in the SCI rats treated with a CDK inhibitor − including decreased immunoreactivity of Iba-1, CD11b and p22PHOX. Together, these data suggest that suppression of chronic inflammation by cell cycle inhibition may account, at least in part, for the progressive tissue loss after SCI. The results also suggest that persistent cell cycle activation after injury may reflect a positive feedback loop that can be interrupted with sub-acute cell cycle inhibitor administration.
Cell cycle proteins are also expressed in other cell types of the CNS [53, 54], such as oligodendrocytes and infiltrating Schwann cells, which contribute to myelin repair in the injured spinal cord . We recently reported increases in the myelinated white matter area and expression of myelin basic protein in flavopiridol-treated injured rats . However, it remains unclear whether cell cycle inhibition increases remyelinated axons by oligodendrocytes and Schwann cells, or reduces chronic progressive demyelination. We showed CDK4 and E2F5 are highly expressed in the central lesion areas where astrocytes are absent but p75+ Schwann cells have infiltrated [24, 38]. Postnatal Schwann cell proliferation has been known to be strictly and uniquely dependent on CDK4 . However, further investigation is required to elucidate the mechanisms by which cell cycle inhibition modulates myelination after SCI.
CR8 exhibits a 50-fold higher potency than roscovitine in different cell lines, possibly owing its added efficacy to more potent inhibition of CDKs 1, 2, 5, 7 and 9, and increased solubility, cell permeability and enhanced intracellular stability [36, 57]. More recently, we reported that CR8 at a single dose 20 times less than roscovitine [29, 30] provides superior neuroprotection to the parent compound . Given the increased potency and efficacy of CR8 as compared to earlier purine analog types of CDK inhibitors, this drug was used systemically in the present study. CR8 treatment limited sustained upregulation of cell cycle protein expression, as well as chronic reactive astrocytosis and microglial activation. Significantly reduced lesion volume and improved long-term functional recovery were also observed, suggesting that chronic cell cycle activation may contribute to secondary injury and expansion of the lesion site after SCI.
In summary, we provide evidence that SCI is accompanied by a prolonged, sustained upregulation of cell cycle-related protein expression that may contribute to the development of glial scar formation, chronic inflammation and progressive tissue loss. Blockade of cell cycle pathways by a CDK inhibitor significantly reduces delayed upregulation of cell cycle proteins, limits astrogliosis and chronic inflammation, and subsequent lesion progression, with marked improvement in functional recovery. Thus, sustained cell cycle dysregulation may contribute to the chronic progressive secondary injury after SCI.