The behavioral and biochemical effects of BDNF containing polymers implanted in the hippocampus of rats

Abstract

Brain-derived neurotrophic factor (BDNF) is closely linked with neuronal survival and plasticity in psychiatric disorders. In this work, we engineered degradable, injectable alginate microspheres and non-degradable, implantable poly(ethylene vinyl acetate) matrices to continuously deliver BDNF to the dorsal hippocampus of rats for two days or more than a week, respectively. The antidepressant-like behavioral effects of BDNF delivery were examined in the Porsolt forced swim test. Rats were sacrificed 10days after surgery and tissue samples were analyzed by western blot. A small dose of BDNF delivered in a single infusion, or from a two-day sustained-release alginate implant, produced an antidepressant-like behavior, whereas the same dose delivered over a longer period of time to a larger tissue region did not produce antidepressant-like effects. Prolonged delivery of BDNF resulted in a dysregulation of plasticity-related functions: increased dose and duration of BDNF delivery produced increased levels of TrkB, ERK, CREB, and phosphorylated ERK, while also producing decreased phosphorylated CREB. It is evident from this work that both duration and magnitude of BDNF dosing are of critical importance in achieving functional outcome.

Figure 1. Schematic describing EVAc and alginate biomaterial construction

Porous EVAc matrices were constructed by dissolving polymer with drug and an inert dispersant in organic solvent. This mixture was poured onto a chilled aluminum mold and the solvent was subsequently evaporated. Biodegradable alginate microspheres were created by adding cross-linker to an emulsified alginate-drug solution.

Figure 2. Polymeric biomaterials were designed to deliver a sustained dose of BDNF to the hippocampus

(a) Controlled release measurements of BDNF from alginate and EVAc biomaterials. The majority of BDNF was released from alginate materials over a period of 1–2 days; EVAc materials continued releasing BDNF for 7 days. Release is expressed as a fraction of total protein released for alginate and as fraction total theoretical loading for EVAc. (b) Image of an EVAc disc implanted into the rat hippocampus. Rats received solid EVAc matrices and were sacrificed 10 days later. The EVAc implant (I) reached the target location of the DG/CA1 regions of the dorsal hippocampus. Nissl stain revealed only minimal tissue damage surrounding the implant location (arrowhead) (c) Light microscope image of alginate microspheres. Microspheres were of round, uniform morphology, with an average diameter of 38 µm. Scale bar = 50µm.

Figure 3. PC12-TrkB response to BDNF

(a) Serum deprived PC12-TrkB cells extended neurites in response to free BDNF. (b–d) However, no neurite extension was observed for non-treated or blank-material controls. Neurite extension was observed in the presence of polymer-encapsulated and polymer-released BDNF (50ng/ml).. (i) Neurite length was significantly greater in BDNF treated cells when compared to non-treated cells, regardless of the source of BDNF (error bars represent standard deviation).

Figure 4. Effect of BDNF on behavioral outcomes in the forced swim test for varying delivery methods

(a) No differences in swimming, climbing or immobility behavior were observed for control groups that received saline infusions, blank microsphere or blank EVAc implants (one-way ANOVA, p=0.95), indicating that the vehicle itself does not alter behavior. (b) A single, LD infusion of BDNF and LD microsphere implants produced antidepressant-like effects, as evident by a decrease in immobility compared to pooled control (p=0.0023 and 0.0022, respectively). However, rats that received LD, MD or HD matrix implants did not behave differently than control rats (p=0.95, 0.73 and 0.58, respectively). (c,d) LD infusions of BDNF and LD microsphere implants produced increased swimming behavior compared to control rats (p=0.0012 and 0.039, respectively). However, rats that received LD, MD or HD matrices did not behave differently than control rats (p=0.79, 0.74 and 0.44, respectively). No differences were observed in climbing behavior. Statistical comparisons were performed for each dosing group compared to pooled, no-dose controls.

Figure 5. Activation of the TrkB-ERK-CREB signaling pathway as a function of BDNF delivery

LD infusion, LD alginate, LD EVAc, MD EVAc, and HD EVAc are ranked in order of increasing dose and duration of BDNF exposure, with material controls represented by a value of 1. Total levels of TrkB, ERK, and CREB increased for higher dose and duration of BDNF exposure (p = 0.0036, 0.00030, and 0.022, respectively). Phosphorylated ERK increased and phosphorylated CREB decreased for higher dose and duration of BDNF exposure (p = 0.023 and 0.028, respectively), whereas phosphorylated TrkB was unchanged (p = 0.33). The slope of each signficant relationship is depicted by a dashed line appearing above the data.