Intracerebral Injection of “Parenchygel” in Rat Intracerebral Hemorrhage Accelerated Recover and Recruits Endogenous Microglia/Macrophages

Introduction: : Approximately 100,000 people in the United States experience intracerebral hemorrhage (ICH) each year, leading to a mortality rate of 40% - 50%. ICH is split into two phases. Initial primary brain damage is mechanical and irreversible. Secondary brain damage occurs days to weeks after the primary injury, where neuroinflammation increases and inflammatory (M1) microglia are recruited to the injury site. Toward the goal of creating a treatment for secondary brain damage, we have developed a brain tissue-inspired hydrogel depot to mimic native brain parenchyma and locally deliver recombinant cytokines (IL-4 and IL-10) to modulate the neuro- inflammation. This delivery platform is dubbed “Parenchygel”, a brain tissue mimicry of the delivery vehicle. Parenchygel is comprised of a formulation of hyaluronic acid, poly (ethylene glycol), heparin, and poly(lactic-co-glycolic acid) PLGA nanoparticles, which can stabilize and deliver cytokines. Here, the limitations of recombinant cytokine therapy (i.e., short half-life and systemic toxicity) are overcome by using an intracerebral injection of this hydrogel, which is loaded with cytokine and provides for controlled and localized drug delivery.
We hypothesized that hydrogel-mediated delivery of recombinant cytokines to rat ICH would improve functional recovery by recruiting alternatively activated microglia/macrophages to the site of injury. To test this hypothesis, we induced ICH in healthy rats via double-blood injection and evaluated the hydrogel-cytokine therapeutic, administered 24 hours later. The therapeutic study included four groups - a control group with no intervention, a surgery control (ICH + hematoma evacuation), a carrier control (hydrogel), and a therapeutic arm (hydrogel + cytokines).

Materials and

Methods: : To establish the Parenchygel platform, methacrylated hyaluronic acid, poly (ethylene glycol) dimethacrylate, thiolated heparin, and acid-capped Poly(lactic-co-glycolic) (PLGA) nanoparticles were mixed and chemically crosslinked (LAP photoinitiator, 365 nm light exposure, 10 minutes). To evaluate Parenchygel platform as an immunotherapy for secondary brain damage caused by ICH, a therapeutic study assessed the recruitment of microglia/macrophages in a rat double-blood injection stroke model. All the rats received a total injection of 50 μL of blood, obtained from the tail vein. Rats in the surgical, carrier, and therapeutic groups had the hematoma evacuated the next day. Rats in the carrier and therapeutic group received a 20 μL injection of Parenchygel. All the rats were monitored for 34 days using the modified stroke scoring system (mNSS), which monitored their sensory, motor, balance, and reflexes. Neurological scoring, mNSS, was conducted on 1, 3, 5, 7, 8, 13, 20, 27, and 34 days post-surgery. To assess the formation of a stroke, the brains of the untreated control group were sectioned and H&E stained. The success of the hydrogel injection was measured by sectioning and H&E staining the carrier and therapeutic groups. Neuron morphology was further evaluated with cresyl violet staining. The recruitment of microglia/macrophages was assessed using immunostaining for Iba1 (activated microglia/macrophages), inflammatory markers (CD 86, iNOS), and anti-inflammatory (CD206, Arginase-1) in brain tissue sections obtained at the 34-day endpoint.

Results, Conclusions, and Discussions:: A therapeutic study determined the ability of Parenchygel to promote functional recovery by recruiting alternatively activated microglial/macrophages. Results from the mNSS scoring demonstrated the ability to induce ICH in rats, shown by the control group (no evacuation-no treatment) in Figure 1a, where stroke scores increased to a score of four. In contrast, the therapeutic group (evacuation + hydrogel + cytokine) showed a statistically significant reduction in mNSS scoring 3 days after, where the mNSS score returned to baseline (score of zero) (p-value:>0.01). Further investigation by a histopathologist revealed that the tissue showed no increase in neuroinflammation or tissue damage (Figure 1b). Additionally, the histology showed a recruitment of cells around the hydrogel injection (Figure 1b). Immunofluorescence was used to identify these cells, specifically Iba1+ for microglia/macrophages, anti-inflammatory markers (Arginase-1, CD 206), and inflammatory markers (iNOS, CD 86). Results from the immunofluorescence in Figure 2 showed activated microglia/macrophages surrounding the injection of hydrogel loaded with cytokines. Furthermore, the cells showed expression of both anti-inflammatory and inflammatory markers. The combination of histology and immunofluorescence showed microglia/macrophages recruited to the hydrogel injection site . While the mNSS scoring showed a functional recovery in rats receiving hydrogel injection and recombinant cytokines. This data supports that the hydrogel loaded with cytokines is improving functional recovery and recruiting microglia/macrophages.
This study showed that Parenchygel can be utilized to locally intervene post-ICH and stimulate the immune system. Parenchygel-mediated delivery of recombinant IL-4 and IL-10 not only accelerated motor and sensory recovery in rats but also stimulated and recruited microglia/macrophages to the site of injury. Our data suggests that these recruited cells adopt a hybrid M1/M2 phenotype, which could contribute to post-ICH neurological recovery. Overall, our study contributes to the development of a novel platform for immune therapy of brain inflammation, with translational potential for secondary brain damage post-ICH.

Acknowledgements and/or References (Optional):: Funding is gratefully acknowledged from the OUHSC-IBEST collaborative research program (OU-Norman) and the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health under award number 1R35GM150970 . Microscopy data collection was performed at the Samuel Roberts Noble Microscopy Laboratory, an OU core facility supported by the Vice President for Research and Partnerships