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Additional File 1 Supplementary Fig. S1. Genotyping of RIPK1 and RIPK3 deficient mice and proof of neuronal specific RIPK1 knock-out in RIPK1flox/floxCamk2CreERT2 mice. a. Neuron specific RIPK1 deficient mice used for experiments were heterozygous for Camk2CreERT2 and homozygous for the floxed RIPK1 allele. Littermate controls were also homozygous for the floxed RIPK1 allele, but did not express the Cre recombinase. b. Global RIPK3 deficient mice were homozygous for disrupted allele, while control mice expressed only the wild type gene. c. and d. To demonstrate specific neuron specific RIPK1 deficiency in induced RIPK1flox/floxCamk2CreERT2 mice, we performed immunohistochemistry for RIPK1 and NeuN, a neuronal marker. In the cortex of control mice RIPK1 was almost exclusively expressed in neurons (upper panels), while in induced RIPK1flox/floxCamk2CreERT2 mice RIPK1 staining was significantly reduced (lower panels) to 20% of baseline (d). Additional File 1 Supplementary Fig. 2 Body weight and physical condition after experimental TBI. a. and b. Weight after TBI. Animals recovered from weight loss directly after trauma within one week after injury; in the following observation period weight constantly increased. No differences were detected between CCI and sham-operated animals in the RIPK1 (a) and RIPK3 (b) groups. c. and d. General health score to assess recovery. All animals��� general condition transiently worsened in the perioperative phase with a peak at day 1 after TBI, but returned to baseline within one week. There was no difference between groups, c. RIPK1, d. RIPK3. Data are presented as mean �� SD; n = 5 for sham, n = 8���10 for TBI. Additional File 1 Supplementary Fig. 3. Individual lesion volume progression for a. RIP1 and b. RIP3 deficient mice. Additional File 1 Supplementary Fig. 4. RIPK3 deficiency does not affect acute brain injury after TBI. No differences in lesion volume as assessed by histology was detected between RIPK3 knockout mice and C57BL/6 wild type controls at 24 h after TBI. Data are presented as mean �� SD; n = 10. Additional File 1 Supplementary Fig. 5. Lesion volumes by MRI and histology. a. T2-weighted MRI and Nissl stained coronal section three months after injury. Lesion volume was quantified in T2-weighted MRI images as well as Nissl stained sections obtained in the same animals three months post injury. b. Correlation analysis of lesion volumes assessed by histomorphometry or T2-weighted MRI revealed a strong positive correlation between both methods. Differences in measured lesion size are due to shrinking of tissue in the fixation process, with a shrinking factor of 0.77 �� 0.23. c.���f. Quantification of lesion volume (c. RIPK1, d. RIPK3) as well as hippocampal damage (e. RIPK1, f. RIPK3) using both methods. Data are presented as mean �� SD; n = 8���10 for TBI. Pearson correlation, Students t-test for parametric and Man-Whitney-Rank-Sum test for non-parametric data were used. *p

The protective properties of the blood-brain barrier (BBB) are conferred by the intricate architecture of its endothelium coupled with multiple specific transport systems expressed on the surface of endothelial cells (ECs) in the brain's vasculature. When the stringent control of the BBB is disrupted, such as following EC damage, substances that are safe for peripheral tissues but toxic to neurons have easier access to the central nervous system (CNS). As a consequence, CNS disorders, including degenerative diseases, can occur independently of an individual's age. Although the BBB is crucial in regulating the biochemical environment that is essential for maintaining neuronal integrity, it limits drug delivery to the CNS. This makes it difficult to deliver beneficial drugs across the BBB while preventing the passage of potential neurotoxins. Available options include transport of drugs across the ECs through traversing occludins and claudins in the tight junctions or by attaching drugs to one of the existing transport systems. Either way, access must specifically allow only the passage of a particular drug. In general, the BBB allows small molecules to enter the CNS; however, most drugs with the potential to treat neurological disorders other than infections have large structures. Several mechanisms, such as modifications of the built-in pumping-out system of drugs and utilization of nanocarriers and liposomes, are among the drug-delivery systems that have been tested; however, each has its limitations and constraints. This review comprehensively discusses the functional morphology of the BBB and the challenges that must be overcome by drug-delivery systems and elaborates on the potential targets, mechanisms, and formulations to improve drug delivery to the CNS.

Igor Khalin,1 Renad Alyautdin,2 Ganna Kocherga,3 Muhamad Abu Bakar1 1Faculty of Medicine and Defence Health, National Defence University of Malaysia, Kuala Lumpur, Malaysia; 2Scientific Centre for Expertise of Medical Application Products, Moscow, Russia; 3Ophthalmic Microsurgery Department, International Medical Center Oftalmika, Kharkiv, UkraineAbstract: Neurodegenerative causes of blindness and deafness possess a major challenge in their clinical management as proper treatment guidelines have not yet been found. Brain-derived neurotrophic factor (BDNF) has been established as a promising therapy against neurodegenerative disorders including hearing and visual loss. Unfortunately, the blood–retinal barrier and blood–cochlear barrier, which have a comparable structure to the blood–brain barrier prevent molecules of larger sizes (such as BDNF) from exiting the circulation and reaching the targeted cells. Anatomical features of the eye and ear allow use of local administration, bypassing histo-hematic barriers. This paper focuses on highlighting a variety of strategies proposed for the local administration of the BDNF, like direct delivery, viral gene therapy, and cell-based therapy, which have been shown to successfully improve development, survival, and function of spiral and retinal ganglion cells. The similarities and controversies for BDNF treatment of posterior eye diseases and inner ear diseases have been analyzed and compared. In this review, we also focus on the possibility of translation of this knowledge into clinical practice. And finally, we suggest that using nanoparticulate drug-delivery systems may substantially contribute to the development of clinically viable techniques for BDNF delivery into the cochlea or posterior eye segment, which, ultimately, can lead to a long-term or permanent rescue of auditory and optic neurons from degeneration. Keywords: brain-derived neurotrophic factor, neurodegeneration, posterior eye segment, inner ear, targeted drug-delivery, nanoparticles

Renad Alyautdin,1 Igor Khalin,2 Mohd Ismail Nafeeza,1 Muhammad Huzaimi Haron,1 Dmitry Kuznetsov31Faculty of Medicine, Universiti Teknologi MARA (UiTM), Sungai Buloh, Selangor, Malaysia; 2Faculty of Medicine and Defence Health, National Defence University of Malaysia (NDUM), Kuala Lumpur, Malaysia; 3Department of Medicinal Nanobiotechnologies, N. I. Pirogoff Russian State Medical University, Moscow, RussiaAbstract: The protective properties of the blood–brain barrier (BBB) are conferred by the intricate architecture of its endothelium coupled with multiple specific transport systems expressed on the surface of endothelial cells (ECs) in the brain's vasculature. When the stringent control of the BBB is disrupted, such as following EC damage, substances that are safe for peripheral tissues but toxic to neurons have easier access to the central nervous system (CNS). As a consequence, CNS disorders, including degenerative diseases, can occur independently of an individual's age. Although the BBB is crucial in regulating the biochemical environment that is essential for maintaining neuronal integrity, it limits drug delivery to the CNS. This makes it difficult to deliver beneficial drugs across the BBB while preventing the passage of potential neurotoxins. Available options include transport of drugs across the ECs through traversing occludins and claudins in the tight junctions or by attaching drugs to one of the existing transport systems. Either way, access must specifically allow only the passage of a particular drug. In general, the BBB allows small molecules to enter the CNS; however, most drugs with the potential to treat neurological disorders other than infections have large structures. Several mechanisms, such as modifications of the built-in pumping-out system of drugs and utilization of nanocarriers and liposomes, are among the drug-delivery systems that have been tested; however, each has its limitations and constraints. This review comprehensively discusses the functional morphology of the BBB and the challenges that must be overcome by drug-delivery systems and elaborates on the potential targets, mechanisms, and formulations to improve drug delivery to the CNS.Keywords: apoE, blood–brain barrier, CNS, drug targeting, liposomes, nanoparticles