Melinda Fitzgerald

Redefining the role of metallothionein within the injured brain: extracellular metallothioneins play an important role in the astrocyte-neuron response to injury

A number of intracellular proteins that are protective after brain injury are classically thought to exert their effect within the expressing cell. The astrocytic metallothioneins (MT) are one example and are thought to act via intracellular free radical scavenging and heavy metal regulation, and in particular zinc. Indeed, we have previously established that astrocytic MTs are required for successful brain healing. Here we provide evidence for a fundamentally different mode of action relying upon intercellular transfer from astrocytes to neurons, which in turn leads to uptake-dependent axonal regeneration. First, we show that MT can be detected within the extracellular fluid of the injured brain, and that cultured astrocytes are capable of actively secreting MT in a regulatable manner. Second, we identify a receptor, megalin, that mediates MT transport into neurons. Third, we directly demonstrate for the first time the transfer of MT from astrocytes to neurons over a specific time course in vitro. Finally, we show that MT is rapidly internalized via the cell bodies of retinal ganglion cells in vivo and is a powerful promoter of axonal regeneration through the inhibitory environment of the completely severed mature optic nerve. Our work suggests that the protective functions of MT in the central nervous system should be widened from a purely astrocytic focus to include extracellular and intra-neuronal roles. This unsuspected action of MT represents a novel paradigm of astrocyte-neuronal interaction after injury and may have implications for the development of MT-based therapeutic agents.

Angiostatin Formation Involves Disulfide Bond Reduction and Proteolysis in Kringle 5 of Plasmin

Plasmin is processed in the conditioned medium of HT1080 fibrosarcoma cells producing fragments with the domain structures of the angiogenesis inhibitor, angiostatin, and microplasmin. Angiostatin consists of kringle domains 1–4 and part of kringle 5, while microplasmin consists of the remainder of kringle 5 and the serine proteinase domain. Our findings indicate that formation of angiostatin/microplasmin involves reduction of plasmin by a plasmin reductase followed by proteolysis of the reduced enzyme. We present evidence that the Cys461–Cys540 and Cys511–Cys535 disulfide bonds in kringle 5 of plasmin were reduced by plasmin reductase. Plasmin reductase activity was secreted by HT1080 and Chinese hamster ovary cells and the human mammary carcinoma cell lines MCF-7, MDA231, and BT20 but not by the monocyte/macrophage cell line THP-1. Neither primary foreskin fibroblasts, blood monocyte/macrophages, nor macrovascular or microvascular endothelial cells secreted detectable plasmin reductase. In contrast, cultured bovine and rat vascular smooth muscle cells secreted small but reproducible levels of plasmin reductase. Reduction of the kringle 5 disulfide bonds triggered cleavage at either Arg529-Lys530 or two other positions C-terminal of Cys461 in kringle 5 by a serine proteinase. Plasmin autoproteolysis could account for the cleavage, although another proteinase was mostly responsible in HT1080 conditioned medium. Three serine proteinases with apparent M r of 70, 50, and 39 were purified from HT1080 conditioned medium, one or more of which could contribute to proteolysis of reduced plasmin.

Early Events of Secondary Degeneration after Partial Optic Nerve Transection: An Immunohistochemical Study

Secondary degeneration in the central nervous system involves indirect damage to neurons and glia away from the initial injury. Partial transection of the dorsal optic nerve (ON) results in precise spatial separation of the primary trauma from delayed degenerative events in ventrally placed axons and parent somata. Here we conduct an immunohistochemical survey of secondary cellular changes in and around axons and their parent retinal ganglion cell (RGC) somata during the first 3 days after a restricted, dorsal ON transection. This is before the secondary loss of RGCs and axons projecting through the uninjured, ventral portion of the ON. Within 5 min, manganese superoxide dismutase (MnSOD; a marker of oxidative stress) co-localizes within the astrocytic network across the entire profile of the ON. Secondary astrocyte hypertrophy of immunofluorescent labeling was evident from 3 h, with sustained increases in myelin basic protein immunoreactivity across the nerve by 24 h. Increases in NG-2-positive oligodendrocyte precursor cells, ED-1-positive activated microglia/macrophages, and Iba1-positive reactive resident microglia/macrophage numbers were only seen in ON vulnerable to secondary degeneration by 3 days. Changes within RGC somata exclusively vulnerable to secondary degeneration were detected at 24 h, as evidenced by increases in MnSOD immunoreactivity, followed by increases in c-jun immunoreactivity at 3 days. Treatment with the voltage-gated calcium channel blocker lomerizine did not alter any measured outcome. We conclude that oxidative stress spreading via the astrocytic network and from injured axons to parent RGC somata is an early event during secondary degeneration, and containment is likely to be required in order to prevent further damage to the nerve.

Multimodal analysis of PEI-mediated endocytosis of nanoparticles in neural cells.

Polymer nanoparticles are widely used as a highly generalizable tool to entrap a range of different drugs for controlled or site-specific release. However, despite numerous studies examining the kinetics of controlled release, the biological behavior of such nanoparticles remains poorly understood, particularly with respect to endocytosis and intracellular trafficking. We synthesized polyethylenimine-decorated polymer nanospheres (ca. 100-250 nm) of the type commonly used for drug release and used correlated electron microscopy, fluorescence spectroscopy and microscopy, and relaxometry to track endocytosis in neural cells. These capabilities provide insight into how polyethylenimine mediates the entry of nanoparticles into neural cells and show that polymer nanosphere uptake involves three distinct steps, namely, plasma membrane attachment, fluid-phase as well as clathrin- and caveolin-independent endocytosis, and progressive accumulation in membrane-bound intracellular vesicles. These findings provide detailed insight into how the intracellular delivery of nanoparticles is mediated by polyethylenimine, which is presently the most commonly used nonviral gene transfer agent. This fundamental knowledge may also assist in the preparation of next-generation nonviral vectors.

Secondary Retinal Ganglion Cell Death and the Neuroprotective Effects of the Calcium Channel Blocker Lomerizine

PURPOSE After partial optic nerve (ON) injury, intact retinal ganglion cells (RGCs) undergo secondary death, but the topographic distribution of this death is unknown, and it is unclear which cell death pathways are involved. Although the calcium channel blocker lomerizine reduces RGC death after partial ON injury, it is unknown whether this drug alleviates necrotic or apoptotic death. METHODS The dorsal ON was transected in adult Piebald-Virol-Glaxo (PVG) rats, and the site of secondary RGC death was determined using anterograde and retrograde DiI tracing. RGC death was assessed at 2 and 3 weeks. Retrograde tracing with fluorogold injected into the superior colliculus 3 days before euthanatization was used to identify RGCs undergoing secondary death. Overall cell loss was quantified using betaIII-tubulin immunohistochemistry. Lomerizine (30 mg/kg, oral) or vehicle was given twice daily, and retinal wholemounts were analyzed for necrotic morphology (nucleic acid stain) or anticleaved caspase-3 expression at 2 and 3 weeks. RESULTS Ventral retina was identified as the site of secondary RGC death, and central and dorsal retinae were defined as sites of both primary and secondary death. Overall RGC loss occurred by 2 weeks in central and ventral retina (P < 0.05) and by 3 weeks in dorsal retina (P < 0.05). Secondary RGC death was characterized mainly by necrotic morphology, with caspase-3 expression in some RGCs. Lomerizine reduced secondary necrosis at 2 weeks and secondary caspase-3 expression at 3 weeks. CONCLUSIONS Lomerizine had differential effects on necrotic and apoptotic death with time, but its inability to completely prevent secondary death suggests that full neuroprotection will require combinatorial treatments.

Near infrared light reduces oxidative stress and preserves function in CNS tissue vulnerable to secondary degeneration following partial transection of the optic nerve.

Traumatic injury to the central nervous system (CNS) is accompanied by the spreading damage of secondary degeneration, resulting in further loss of neurons and function. Partial transection of the optic nerve (ON) has been used as a model of secondary degeneration, in which axons of retinal ganglion cells in the ventral ON are spared from initial dorsal injury, but are vulnerable to secondary degeneration. We have recently demonstrated that early after partial ON injury, oxidative stress spreads through the ventral ON vulnerable to secondary degeneration via astrocytes, and persists in the nerve in aggregates of cellular debris. In this study, we show that diffuse transcranial irradiation of the injury site with far red to near infrared (NIR) light (WARP 10 LED array, center wavelength 670 nm, irradiance 252 W/m(-2), 30 min exposure), as opposed to perception of light at this wavelength, reduced oxidative stress in areas of the ON vulnerable to secondary degeneration following partial injury. The WARP 10 NIR light treatment also prevented increases in NG-2-immunopositive oligodendrocyte precursor cells (OPCs) that occurred in ventral ON as a result of partial ON transection. Importantly, normal visual function was restored by NIR light treatment with the WARP 10 LED array, as assessed using optokinetic nystagmus and the Y-maze pattern discrimination task. To our knowledge, this is the first demonstration that 670-nm NIR light can reduce oxidative stress and improve function in the CNS following traumatic injury in vivo.

Myelin sheath decompaction, axon swelling, and functional loss during chronic secondary degeneration in rat optic nerve

PURPOSE To examine chronic changes occurring at 6 months following partial optic nerve (ON) transection, assessing optic axons, myelin, and visual function. METHODS Dorsal ON axons were transected, leaving ventral optic axons vulnerable to secondary degeneration. At 3 and 6 months following partial transection, toluidine-blue stained sections were used to assess dimensions of the ON injury site. Transmission electron microscopy (TEM) images of ventral ON were used to quantify numbers, diameter, area, and myelin thickness of optic axons. Immunohistochemistry and fluoromyelin staining were used to assess semiquantitatively myelin protein, lipids in ventral ON, and retinal ganglion cells (RGCs) in midventral retina. Visuomotor function was assessed using optokinetic nystagmus. RESULTS Following partial ON transection, optic axons and function remained disrupted at 6 months. Although ventral ON swelling observed at 3 months (P ≤ 0.05) receded to normal by 6 months, ultrastructurally, myelinated axons remained swollen (P ≥ 0.05), and myelin thickness increased (P ≤ 0.05) due to loosening of lamellae and an increase in the number of intraperiodic lines. Axons with decompacted myelin persisted and were distinguished as having large axonal calibers and thicker myelin sheaths. Nevertheless, progressive loss of myelin lipid staining with fluoromyelin was seen at 6 months. Despite no further loss of ventral optic axons between 3 and 6 months (P ≥ 0.05), visuomotor function progressively declined at 6 months following partial transection (P ≤ 0.05). CONCLUSIONS Continued decompaction of myelin, altered myelin structure, and swelling of myelinated axons are persistent features of the chronic phases of secondary degeneration and likely contribute to progressive loss of visual function.

Matrix metalloproteinases can facilitate the heparanase-induced promotion of phenotypic change in vascular smooth muscle cells

Previous studies from this laboratory have shown that degradation of heparan sulphate proteoglycan by both living macrophages and macrophage lysosomal heparanase induces phenotypic change of vascular smooth muscle cells (SMC) from a high volume fraction of myofilaments (V(v)myo) to a low V(v)myo [Campbell et al. Exp Cell Res 1992; 200: 156-167]. The aim of this study was to determine whether matrix metalloproteinase (MMP) activity is also involved in the induction of SMC phenotypic change by macrophages. A specific inhibitor of MMPs (BB94) was able to block macrophage-induced SMC phenotypic change and subsequent DNA synthesis in freshly dispersed SMC seeded in primary culture at confluent density. The inhibitor did not block these SMC changes when SMC were seeded at low density without macrophages nor did it block heparanase activity directly. We also determined whether heparanase and MMP activities are upregulated together in vivo. Artery homogenates were analysed in a heparanase enzyme assay and for MMPs using zymograms. Increased heparanase activity was observed 3-14 days following balloon catheter injury of rabbit carotid arteries, and returned to control levels 6 weeks after injury. Active MMP2 was induced with heparanase after injury. MMP9 induction was also apparent 6 h after injury. Immunohistology on sections of these arteries showed the presence of MMP1, 2, 3 and 9 with these MMPs being strongly induced in the intima 7 days after balloon catheter injury. Both heparanase and MMP activities were also present in human end-stage complex lesions from coronary arteries, carotid endarterectomies and abdominal aortic aneurysms. Because MMPs and heparanase are expressed at the same time, it is possible that MMPs facilitate heparanase activity in promotion of phenotypic modulation of SMC in vivo during neointimal thickening following injury and in atherosclerotic lesions

Red/near-infrared irradiation therapy for treatment of central nervous system injuries and disorders.

Abstract Irradiation in the red/near-infrared spectrum (R/NIR, 630–1000 nm) has been used to treat a wide range of clinical conditions, including disorders of the central nervous system (CNS), with several clinical trials currently underway for stroke and macular degeneration. However, R/NIR irradiation therapy (R/NIR-IT) has not been widely adopted in clinical practice for CNS injury or disease for a number of reasons, which include the following. The mechanism/s of action and implications of penetration have not been thoroughly addressed. The large range of treatment intensities, wavelengths and devices that have been assessed make comparisons difficult, and a consensus paradigm for treatment has not yet emerged. Furthermore, the lack of consistent positive outcomes in randomised controlled trials, perhaps due to sub-optimal treatment regimens, has contributed to scepticism. This review provides a balanced précis of outcomes described in the literature regarding treatment modalities and efficacy of R/NIR-IT for injury and disease in the CNS. We have addressed the important issues of specification of treatment parameters, penetration of R/NIR irradiation to CNS tissues and mechanism/s, and provided the necessary detail to demonstrate the potential of R/NIR-IT for the treatment of retinal degeneration, damage to white matter tracts of the CNS, stroke and Parkinson’s disease.

In vivo Imaging and Biodistribution of Multimodal Polymeric Nanoparticles Delivered to the Optic Nerve

The use of nanoparticles for targeted delivery of therapeutic agents to sites of injury or disease in the central nervous system (CNS) holds great promise. However, the biodistribution of nanoparticles following in vivo administration is often unknown, and concerns have been raised regarding potential toxicity. Using poly(glycidyl methacrylate) (PGMA) nanoparticles coated with polyethylenimine (PEI) and containing superparamagnetic iron oxide nanoparticles as a magnetic resonance imaging (MRI) contrast agent and rhodamine B as a fluorophore, whole animal MRI and fluorescence analyses are used to demonstrate that these nanoparticles (NP) remain close to the site of injection into a partial injury of the optic nerve, a CNS white matter tract. In addition, some of these NP enter axons and are transported to parent neuronal somata. NP also remain in the eye following intravitreal injection, a non-injury model. Considerable infiltration of activated microglia/macrophages occurs in both models. Using magnetic concentration and fluorescence visualization of tissue homogenates, no dissemination of the NP into peripheral tissues is observed. Histopathological analysis reveals no toxicity in organs other than at the injection sites. Multifunctional nanoparticles may be a useful mechanism to deliver therapeutic agents to the injury site and somata of injured CNS neurons and thus may be of therapeutic value following brain or spinal cord trauma.