Hyaluronidase and Hyaluronan Oligosaccharides Promote Neurological Recovery After Intraventricular Hemorrhage

Intraventricular hemorrhage (IVH) in premature infants is a devastating problem that occurs tens of thousands of times each year in the United States alone. Survivors face numerous neurological concerns, including cognitive deficits, cerebral palsy, and hydrocephalus. As neurosurgeons, we are humbled by our inability to do much more than manage spinal fluid diversion. One mechanism that appears to underlie the neurological troubles is white matter injury caused by demyelination. It has been discovered that hyaluronan (HA), a negatively charged glycosaminoglycan polymer, is abundantly found in white matter lesions with IVH, where an HA receptor known as CD44 is overexpressed, oligodendrocyte precursors (OPCs) show arrested maturation, and there is reduced myelination.¹ Because of the growing body of information implicating HA as a potential therapeutic target in IVH, Vinukonda et al² sought to investigate an HA-dependent mechanism as a possible therapeutic target to improve myelination via the administration of hyaluronidase or HA oligosaccharides.

Initial experiments sought to determine how HA might lead to demyelination. Using both preterm human autopsy tissue and a premature rabbit pup model of IVH, Vinukonda et al evaluated the overall levels of HA both immunocytochemically and immunohistochemically and did not find them to be meaningfully different in the IVH and non-IVH brains. However, the synthesis and abundance of one of the enzymes that produces HA were increased in the setting of IVH, whereas the expression of hyaluronidase was not affected. HA works via receptors such as CD44 and TLR2/4. All 3 of these receptors were found to be upregulated after IVH compared with the non-IVH tissue. To understand how myelination was affected by IVH, the rabbit model was used and the abundance of myelin basic protein was assessed. Compared with controls, premature rabbit pups with IVH showed significantly reduced myelin basic protein in the corpus callosum and corona radiata. Interestingly, when hyaluronidase was injected intraventricularly, the expression of myelin basic protein was restored. Ultrastructural evaluation of the brain tissue confirmed a diminished abundance of myelinated axons with IVH compared with controls. Importantly, hyaluronidase did not inhibit astrogliosis, nor did it affect myelination or astrogliosis in rabbit pups without IVH.

Because HA inhibits OPC maturation, Vinukonda et al tested the hypothesis that hyaluronidase administration intraventricularly could resurrect it. First, hyaluronidase did not notably affect the abundance of OPCs in either IVH or non-IVH specimens. However, the abundance of more mature OPCs was negatively affected by IVH and was restored with the administration of hyaluronidase. HA works via its receptors to initiate a pathway that is proinflammatory. To test this, the investigators evaluated for the abundance of activated microglia after IVH and treated with hyaluronidase (Figure). The number of activated microglia was indeed increased after IVH, a phenomenon that was reversed by hyaluronidase treatment. The expression of the proinflammatory cytokines tumor necrosis factor-α and interleukin-1β was increased after IVH but reduced after hyaluronidase treatment. To clinically evaluate hyaluronidase treatment, Vinukonda et al performed neurobehavioral evaluations on preterm rabbit pups. The pups treated with vehicle after IVH demonstrated significant functional difficulties across a battery of neurobehavioral tests. Notably, in hyaluronidase-treated preterm pups, scores in these tests were significantly improved. Hyaluronidase treatment alone had no negative impact on normal pups.

Figure: Hyaluronidase treatment reduced microglia density, cytokines, CD44, and TLR4 in rabbits with intraventricular hemorrhage (IVH). A and B, representative immunofluorescence of Iba1 and CD11b is shown in the corona radiata of pups at day 3 as indicated. Sytox staining was used to label nuclei. Insets show high-magnification views of the boxed area in the image. Both Iba1_and CD11B_microglia were higher in number in pups with IVH compared with controls without IVH. Hyaluronidase treatment reduced their density. Scale bars: 50 μm. C, tumor necrosis factor-α and interleukin-1β mRNA expression was elevated in IVH compared with controls without IVH at day 3, and hyaluronidase treatment restored the level. Interferon-γ gene expression was similar in the 3 groups. D, CD44 mRNA expression was higher in IVH compared with controls without IVH at both days 3 and 7, and hyaluronidase treatment restored the level. TLR4 gene expression was elevated in pups with IVH at day 3 but not at day 7. Hyaluronidase treatment reduced TLR4 levels. TLR2 levels were not significantly changed in the 3 sets of pups at days 3 and 7. E, Western blot analyses for TLR4 on the forebrain lysate of rabbit forebrain at day 3 are shown as indicated. Note that hyaluronidase treatment reduces TLR4 protein expression in pups with IVH. Data are mean ± SEM (n = 5 each group). *P < .05, **P < .01, ***P < .001(pups with vs without IVH); #P < .05, ##P < .01, ###P < .001 (vehicle- vs hyaluronidase-treated pups with IVH). Republished with permission of Society for Neuroscience, from Vinukonda G, Dohare P, Arshad A, et al. Hyaluronidase and hyaluronan oligosaccharides promote neurological recovery after intraventricular hemorrhage. J Neurosci. 2016;36(3):872-889; permission conveyed through Copyright Clearance Center, Inc.

HA is known to bind to the heavy chain (HC) of the glycoprotein inter-α-inhibitor in a proinflammatory environment; the product of this binding (HC-HA) further exacerbates inflammation. The enzyme TSG-6 catalyzes this process. Thus, the investigators sought to determine the abundance of HC-HA complexes and the presence of activity of TSG-6 after IVH. Indeed, HC-HA levels were increased in human preterm IVH autopsy specimens compared with controls in specific brain regions. TSG-6 activity was found to be correspondingly elevated. In previous work, these investigators established a mechanism by which they could consume the HC via the introduction of a HA oligosaccharide to prevent the formation of HC-HA.³ To test this as a potential treatment in IVH, HA oligosaccharides (HA10) were administered. HA10 had an effect similar to that of hyaluronidase in reducing activated microglial density and the abundance of proinflammatory cytokines. Myelination was significantly higher in HA10-treated IVH preterm pups compared with vehicle-treated controls, whereas astrogliosis was unaffected. Finally, neurobehavioral testing showed that HA10-treated pups performed superiorly to controls.

Vinukonda et al have taken on an important but overlooked disease, IVH. We are in an era when we as neurosurgeons are more than ever adding things to the brain rather than simply removing tissue from the brain or altering its plumbing. This elegant study demonstrated both mechanistically and clinically the possibility of targeting HA in the treatment of IVH. As these investigators point out, hyaluronidase treatment seems to be promising. It has already been used therapeutically within the cerebrospinal fluid in the developing world to treat children and adults with tuberculous meningitis and hydrocephalus. However, HA has numerous functions (modulation of AMPA receptor mobility and L-type calcium channels), and hyaluronidase could interfere with them, causing important side effects. The field seems ripe for a clinical trial, especially with a disease that, despite its abundance, has no meaningful treatments and has such devastating consequences.