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Intranasal delivery of interleukin-4 attenuates chronic cognitive deficits via beneficial microglial responses in experimental traumatic brain injury

Traumatic brain injury (TBI) is commonly followed by long-term cognitive deficits that severely impact the quality of life in survivors. Recent studies suggest that microglial/macrophage (Mi/MΦ) polarization could have multidimensional impacts on post-TBI neurological outcomes. Here, we report that repetitive intranasal delivery of interleukin-4 (IL-4) nanoparticles for 4 weeks after controlled cortical impact improved hippocampus-dependent spatial and non-spatial cognitive functions in adult C57BL6 mice, as assessed by a battery of neurobehavioral tests for up to 5 weeks after TBI. IL-4-elicited enhancement of cognitive functions was associated with improvements in the integrity of the hippocampus at the functional (e.g., long-term potentiation) and structural levels (CA3 neuronal loss, diffusion tensor imaging of white matter tracts, etc.). Mechanistically, IL-4 increased the expression of PPARγ and arginase-1 within Mi/MΦ, thereby driving microglia toward a global inflammation-resolving phenotype. Notably, IL-4 failed to shift microglial phenotype after TBI in Mi/MΦ-specific PPARγ knockout (mKO) mice, indicating an obligatory role for PPARγ in IL-4-induced Mi/MΦ polarization. Accordingly, post-TBI treatment with IL-4 failed to improve hippocampal integrity or cognitive functions in PPARγ mKO mice. These results demonstrate that administration of exogenous IL-4 nanoparticles stimulates PPARγ-dependent beneficial Mi/MΦ responses, and improves hippocampal function after TBI.

IL-4 protein-loaded liposomes were evaluated by using dynamic light scattering. The concentration of IL-4 protein in the liposomes was examined by the bicinchoninic protein determination kit (Thermo Fisher Scientific, Pittsburgh, PA, USA).
Mice were randomly assigned to receive intranasal administration of vehicle or IL-4 (50 μg/kg body weight) nanoparticles under anesthesia, starting at 6 h and then once daily on days 1-7 and once weekly on day 14, 21, and 28 after CCI, as described. 2 Nanoparticle-packed IL-4 was diluted to a concentration of 0.1 μg/μl, and five drops (~ 2 μl/drop) of the vehicle or diluted IL-4 nanoparticles were pipetted into each nostril with an interval of 2 min between drops. Intranasal administration at this dose is known to result in significant elevation in IL-4 concentration in various brain regions, including the cortex, striatum, corpus callosum, and external capsule, for up to 12-24 h. 2 This intranasal IL-4 treatment regimen has been shown improve long-term outcomes in mouse models of focal cerebral ischemia 2 and TBI 1 .

Behavior tests
Morris water maze: The Morris water maze test was carried out daily for three days immediately before CCI and then daily from days 22 to 27 after CCI. 3 Briefly, a square Plexiglas platform (11 cm × 11 cm) was submerged 1.5 cm under the water's surface in the center of the north-west quadrant of a circular pool (diameter: 109 cm; depth: 33±0.5 cm). The water was maintained at 20±1℃ and non-toxic white tempera paint was added in. Prominent spatial cues were arranged around the pool. The mouse was placed in one of the quadrants without the platform and allowed to swim 60 seconds to find the platform, and then placed on the platform for 30 seconds to reinforce learning at the end of each trail. This test was performed 3 days before CCI to habituate the mice to swimming. Mice that failed to swim or had a bias towards a particular quadrant were excluded from the experiment. The average time spent finding the platform during three trials was quantified as a measure of spatial learning from days 22 to 26 after CCI. On the 27 th day after CCI, the platform was removed, and the mouse was placed in the pool for a single 60-second-long probe trail. The time spent swimming in the target quadrant where the platform had been located previously was recorded as 'spatial memory' and expressed as a percentage of total testing time. Average swim speeds were recorded every day to assess gross motor skills, and mice were excluded if unable to swim the three days before CCI.
Novel object recognition: The novel object recognition test was performed 15 and 29 days after CCI. 4 Mice were placed in the center of the arena for 10 minutes to gain familiarity with two identical, non-appetitive objects placed in two corners (8 cm from adjacent corner walls). Following the pre-test phase, the mouse was placed in the home cage for one hour. For the novel object test phase, one original object was replaced by a novel object (both objects were consistent in height and volume but different in shape), and the mouse was placed back into the arena for 3 minutes. Novel object exploration was defined when the mouse approached an object with its snout (snout within a 2-cm perimeter around the object). The time spent exploring new objects was recorded. The exploration time (%) [(exploration time with novel object / total exploration time) × 100] and the discrimination index [(exploration time with novel object − exploration time with familiar object)/ total exploration time] were calculated, respectively.
Passive avoidance test: The passive avoidance test was performed 35 days after CCI. 5 The chamber was divided into a lightroom and a darkroom, with a gate between the two. Stainless steel grids were placed on the floor of the darkroom to produce a mild foot shock. In the training phase, the gate was opened as the mouse was placed in the lightroom, and the mouse was allowed to enter the darkroom. Once the mouse was entirely in the darkroom, the gate was closed, and a foot shock (0.25 mA, 2s) was delivered. The latency until the animal crossed into the darkroom was recorded. One day after training, retention tests were performed to evaluate fear memory. Each mouse was placed in the lightroom with the gate opened, and the latency until entry into the darkroom from the lightroom was recorded (up to 300s). No electrical stimulation was applied in the test period.
Fluorescence images were taken on a confocal microscope (Fluoview FV1000, Olympus). Images were randomly captured in each mouse in the perilesional CA1, CA3, and DG. The number of neurons, microglia and myelin debris were manually counted in ImageJ software (NIH) by an investigator blinded to experimental grouping.

Diffusion tensor imaging (DTI)
DTI was used to evaluate hippocampal white matter integrity after TBI. 1 At 35 days after TBI, mice were euthanized in a CO2 chamber and perfused with normal saline, followed by 4% PFA.
Ex vivo brains within intact skulls were post-fixed in 4% PFA solution overnight at 4°C and then transferred to PBS before scanning. Magnetic resonance imaging (MRI) was performed using a Bruker AV3HD 11.7 Tesla/89mm vertical-bore microimaging system equipped with a Micro2.5 gradient set capable of 1500mT/m, ParaVision 6.0.1, and a 20 mm quadrature RF resonator (Bruker Biospin, Billerica, MA). Images of 25 contiguous coronal 0.5-mm slices were obtained with a 16 x 15 mm field of view (FOV), 160 ×160 acquisition matrices, 22 ms echo time (TE), 2800 ms repetition time (TR), 3000 s/mm 2 b-value, and 11.0/5.0 ms /. Every slice was scanned twice, and the mean of two scans was collected and further calculated. This setup has been established for the acquisition of high-resolution DTI scanning in small animals. 1 DTI data were analyzed by using DSI Studio software (http://dsi-studio.labsolver.org/). ROIs encompassing the hippocampus were drawn manually in a blinded manner to outline the fiber tracts between CA1 and CA3. Fractional anisotropy (FA) reflects overall microstructural integrity; mean diffusivity (MD) is an inverse measure of membrane density (generally sensitive to cellularity, edema, and necrosis); radial diffusivity (RD) is generally sensitive to myelin integrity. ROIs in the hippocampus were manually drawn by investigators blinded to experimental grouping, where FA, AD, MD, and RD were determined. Directionally-encoded color (DEC), FA, RD, and 3D-reconstruction maps were generated by DSI Studio software.

Proteomic array analysis
Hippocampi harvested from the TBI hemisphere were used for proteomic arrays according to manufacturer's instructions (RayBiotech, Cat No. AAM-INF-1). Briefly, total protein was extracted from hippocampi isolated from TBI or sham-operated animals five days post-surgery, when neuroinflammation is reaching peak in this model. Protein concentrations were quantified by the Bradford method (Bio-Rad, Hercules, USA). A total of 250 µg protein per sample was then loaded to the membrane for detection of inflammatory factors. The optical density of immunoreactive signal was measured using ImageJ software and the expression of inflammatory factors was normalized to the corresponding sham-injured group. Data are presented as fold changes.