(228s) Immobilized BDNF Mimetic Peptides to Combat Secondary Injury Following TBI

Traumatic Brain Injury (TBI) is a debilitating condition for which there currently exists no fully restorative treatment. A primary, mechanical trauma results in instantaneous cell damage and death. Little can be done to address this damage once it has occurred, but a secondary, inflammatory injury cascade persists for weeks and months following the initial insult. Elevated concentrations of free radicals, glutamate induced excitotoxicity, and other comorbidities greatly exacerbate the initial damage and effects of injury. Despite the hostile, complex nature of the secondary injury environment, the timing offers a potential window for treatment. Promising research efforts to protect healthy tissue following TBI have included the delivery of neurotrophic factors, such as brain derived neurotrophic factor (BDNF). BDNF can provide neuroprotection and improve neuron survival following TBI, but it is rapidly cleared in vivo. Recently, short peptides that mimic the activity of native BDNF have been identified. Our laboratory has routinely created functional biomaterials by covalently grafting small molecules and peptides to collagen. We propose to immobilize these BDNF fragment peptides (IKRG) to a polymer matrix for sustained presentation of neurotropic cues at the site of TBI to combat the effects of secondary injury. Herein we present our initial efforts using collagen as a carrier molecule. BDNF fragment peptides were covalently grafted to lysine residues on the collagen backbone via 1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC). A mixture of IKRG peptide, EDC, and collagen is reacted overnight at 4°C, dialyzed to remove un-grafted peptide, lyophilized, and reconstituted at 3mg/mL in 0.02N acetic acid. Peptide grafted collagen was used to treat cultures of cerebellar granule neurons (CGNs) following simulated secondary injury in vitro. CGNs were isolated from day 6 rat pups and plated onto poly-L-lysine coated tissue culture well plates at 2,000,000 cells/mL. Cultures were treated with Cytosine β-D-arabinofuranoside (Ara-C) after 48 hours to limit the proliferation of astrocytes. At DIV 9, secondary injury was modeled by treating cultures with 300µM or 500µM glutamate for one hour after which glutamate containing media was removed. Fresh media containing recovery treatments was added for a period of 24 hours. Immediately following recovery treatments, cellular metabolic activity was evaluated with a MTT assay. Recovery treatments consisted of different ratios of IKRG grafted collagen and native collagen at a net concentration of 0.33mg/mL in culture media. These were compared to untreated and uninjured controls. Following injury with glutamate, CGN cultures demonstrated ~60% reduction in metabolic activity (Untreated 300µM: 45.0 ±13.4; 500µM 33.5 ±11.9). After treatment with native collagen, metabolic activity further decreases (Native Collagen 300µM: 32.9 ±6.2; 500µM 22.2 ±5.5). Treatment with IKRG grafted collagen resulted in a nearly two-fold increase in metabolic activity as compared to native collagen (IKRG-Collagen 300µM: 54.6 ±19.5; 500µM 44.3 ±10.4), but represented only a slight increase as compared to the untreated condition. This suggests that collagen may be having unintended, negative consequences on neuronal viability that is masking the full potential of these IKRG peptides. To realize the full potential of immobilized, BDNF mimetic peptides we are investigating PEG based polymers as a bio-inert alternative for a carrier molecule. Our hope is that immobilization to an alternative polymer will allow us to deliver IKRG peptides without the confounding effects of collagen.