Intracerebral Cytokine Delivery Safely Accelerates Neurological Recovery in a Rat Intracerebral Hemorrhage Model

Introduction: Currently there is an unmet treatment option for secondary brain damage, occurring after an intracerebral hemorrhage (ICH). Toward the goal of intracerebral delivery of disease-modifying therapeutics, we have developed a brain tissue-inspired hydrogel depot to mimic native brain parenchyma, to locally deliver cytokines (IL-4 and IL-10), modulating the immune system to reduce neurological inflammation caused by diseases. Cytokines regulate immune cell phenotype, but their translation is limited by short half-life, instability, and systemic pleiotropy. Local delivery of cytokines within surgically generated voids in the brain parenchyma following hematoma evacuation could overcome the limitations of cytokine therapeutics and enable a new treatment for secondary brain damage improving functional outcomes. Here, we hypothesized that hydrogel loaded with cytokines will improve the functional outcome after the onset of ICH, and evacuation of the hematoma. To test this hypothesis, we ensured the safety of the delivery platform in healthy rats, induced hemorrhagic stroke in a rat model, and evaluated rat recovery following intracerebral (IC) injection of blank hydrogels, or hydrogel + cytokine therapeutic. We developed the delivery platform, named “Parenchygel”, which is an optimized formulation of hyaluronic acid, poly (ethylene glycol), heparin, and poly(lactic-co-glycolic) (PLGA) nanoparticles to stabilize and deliver cytokines. The safety of Parenchygel was assessed in-vivo, locally, and systemically, in healthy rats using a combination of histology, multiplex cytokine assay of blood plasma, and neurological behavior scoring. Efficacy was established in an ICH rat model (double-blood injection), based on neurological behavior scoring (intermediate) and histology (end-point) measures.

Materials and

Methods: Parenchygel was synthesized by covalently crosslinking modified hyaluronic acid, poly(ethylene glycol), heparin, and Poly(lactic-co-glycolic) nanoparticles. A safety study was conducted in healthy rats with the treatment group (Parenchygel, n = 5) and a control group (saline, n = 5). The rats were injected with 20 µl of Parenchygel or saline, respectively, and monitored for 6 weeks. Systemic safety was evaluated with a multiplex cytokine assay (Biorad® Th1 Th2) analyzing inflammatory and anti-inflammatory cytokine concentrations from blood plasma, collected on days 0, 7, 14, and 34. The liver and kidney were stained with hematoxylin and eosin (H&E). The local safety of the hydrogel was evaluated by H&E-stained brain sections. Modified Neurological Severity Score (mNSS) measured neurological deficit. The therapeutic was evaluated in rats induced with ICH, with a control group (no treatment, n =5), surgical intervention (evacuation of hematoma, n = 5), carrier group (Parenchygel, n = 5), and a therapeutic group (Parenchygel, IL-4 and IL-10, n = 5). All the rats received an injection of 50 μL of blood. Rats in the surgical, carrier, and therapeutic group had the hematoma evacuated the next day. Rats in the carrier and therapeutic group received a 20 μL injection of Parenchygel following hematoma evacuation. All the rats were monitored for 6 weeks, and mNSS scoring was conducted on 1, 3, 5, 7, 8, 13, 20, 27, and 34 post-surgeries. To assess the stroke formation, evacuation, and tissue reaction, the brains of all rats were H&E stained. Statistical analysis was performed using GraphPad Prism.

Results, Conclusions, and Discussions: Results and

Discussion: A multiplex assay measured cytokine concentration in healthy rats' blood plasma, and showed no significant difference between groups, additionally, the histology of organs revealed no abnormalities in groups (p-value: < 0.01). Moreover, the mNSS scoring did not indicate any motor, sensory, or reflex neurological consequence as portrayed in Figure 1a. Brain histology confirmed the hydrogel was injected and that there was no visual difference between the control and the treatment group. These experiments support the systemic and local safety of Parenchygel.
Results from the mNSS scoring supported strokes were induced, shown by the control group (no evacuation-no treatment) in Figure 1B where stroke scores increased after induction of ICH. However, the therapeutic group (Evacuation-Hydrogel therapeutic) saw a statistically significant reduction in mNSS scoring 3 days after infarct removal (p-value < 0.01, relative to untreated). Histological analysis of rats with induced stroke and no surgical intervention revealed that hematomas formed in the animals (Figure 2A). While rats who received the evacuation and therapeutic had hydrogel present with no visual immune response or blood clot. The mNSS scoring and preliminary histology support the therapeutics' accelerated recovery.

Conclusions: Our study established the safety of our Parenchygel systemically and locally, characterized by an absence of toxicity or inflammatory response. Our study supports the feasibility of injecting Parenchygel loaded with IL-4 and IL-10 to reduce neurological deficits following ICH and hematoma evacuation at 24 hours. Overall, our study contributes to the development of a novel platform for local immune therapy for hemorrhagic stroke.

Acknowledgements (Optional): Funding is gratefully acknowledged from the OUHSC-IBEST Collaborative Research Program, American Cancer Society IRG2023-1, Harold Hamm foundation, and the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health under award number 1R35GM150970.