Poster Presentation & Flash Talk 46th Annual Meeting of the Fetal and Neonatal Physiological Society 2019

Working towards a novel hydrogel therapy for neonatal stroke (#145)

Bobbi Fleiss 1 2 , Emily Ross-Munro 1 , Kate Firipis 3 4 , James Tran 1 , Madhavi Khore 1 , Richard Williams 5 , David W Walker 1 , Michelle Rank 6 , Mary Tolcos 1
  1. Neurodevelopment in Health and Disease Program, School of Health and Biomedical Sciences, RMIT University, Bundoora, VIC, Australia
  2. Neurodiderot, Hôpital Robert Debré, INSERM UMR 1141, Paris, France
  3. School of Engineering, RMIT University, Bundoora, VIC, Australia
  4. Biofab3D, Aikenhead Centre for Medical Discovery, St Vincent's Hospital, Melbourne, VIC, Australia
  5. School of Medicine, Deakin University, Waurn Ponds, VIC, Australia
  6. Department of Anatomy and Neuroscience, The University of Melbourne, Melbourne, VIC, Australia

Background: Neonatal stroke (NS) leads to permanent neurological deficits and the delay to diagnosis is on average 24 hours. Thus, treatments for NS must target the delayed phases of damage plus stimulate regeneration.

Objectives: To create a biocompatible hydrogel that can be injected directly into stroke tissue to deliver multiple therapeutic agents and restore structural integrity. Also, to characterise a rat model of NS for testing our hydrogel.

Method:  The hydrogel (Fmoc-DIKVAV) was “functionalised” using electrostatic and steric hindrance encapsulation with three molecules: Hi1a to block post-ischemic acidosis-linked cell death; fucoidan to reduce inflammation; and Reg3alpha to stimulate neuroregeneration. We studied in vitro release kinetics over 21 days using ELISA. To induce a (stroke-like) lesion, in postnatal day ten rats we caused a photothrombosis (Rose Bengal, 50mg/kg, IP, 595nm light beam 10 min) within the sensorimotor cortex (Fig.1A). Immunohistochemistry was performed at +6, +24 and +48 hours (all n=6) post-injury for glial fibrillary acidic protein (GFAP; astrocytes), ionised binding adaptor molecule-1 (IBA1; microglia) and cluster of differentiation 68 (CD68; macrophages). We measured lesion volume via H&E (Fig.1B) and studied the size and cell populations in the transition zone, i.e. between the healthy and infarct tissue (Fig.1C).

Results: We successfully functionalised our hydrogel, incorporating fucoidan and delaying the release of Reg3alpha over the days/weeks. GFAP-positive astrocytes became prominent in the transition zone between +6 and +24 hours post-injury. IBA1-positive microglia were found in the core and the transition zone and became ameboid between +6 and +48 hours post-injury (Fig.1D). CD68-positive macrophages were only found in the transition zone, increasingly in number between +24 and +48 hours post-injury (Fig.1E)

Conclusions: We have a biocompatible hydrogel that can deliver multiple drugs in a temporally regulated manner and a model with a reproducible stroke volume for rapid neurotherapeutic testing.

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BF&ERM=joint first-authorship. MR&MT=joint last-authorship.