Exogenous metallothionein-IIA promotes accelerated healing after a burn wound.

Severe injury to the epidermal barrier often results in scarring and life-long functional deficits, the outcome worsening with a number of factors including time taken to heal. We have investigated the potential of exogenous metallothionein IIA (Zn(7)-MT-IIA), a naturally occurring small cysteine-rich protein, to accelerate healing of burn wounds in a mouse model. Endogenous MT-I/II expression increased in basal keratinocytes concurrent with reepithelialization after a burn injury, indicating a role for MT-I/II in wound healing. In vitro assays of a human keratinocyte cell line indicated that, compared with saline controls, exogenous Zn(7)-MT-IIA significantly increased cell viability by up to 30% (p<0.05), decreased apoptosis by 13% (p<0.05) and promoted keratinocyte migration by up to 14% (p<0.05), all properties that may be desirable to promote rapid wound repair. Further in vitro assays using immortalized and primary fibroblasts indicated that Zn7-MT-IIA did not affect fibroblast motility or contraction (p>0.05). Topical administration of exogenous Zn(7)-MT-IIA (2 microg/mL) in vivo, immediately postburn accelerated healing, promoted faster reepithelialization (3 days: phosphate-buffered saline (PBS), 8.9+/-0.3 mm diameter vs. MT-I/II, 7.1+/-0.7 mm; 7 days: PBS 5.8+/-0.98 mm vs. MT-I/II, 3.6+/-1.0 mm, p<0.05) and reduced epidermal thickness (MT-I/II: 45+/-4 microm vs. PBS: 101+/-19 microm, p<0.05) compared with controls. Our data suggest that exogenous Zn(7)-MT-IIA may prove a valuable therapeutic for patients with burns and other skin injuries.

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.

Erythropoietin is both neuroprotective and neuroregenerative following optic nerve transection.

The cytokine hormone erythropoietin (EPO) is neuroprotective in models of brain injury and disease, and protects retinal ganglion cells (RGC) from cell death after axotomy. Here, we assessed EPO's neuroprotective properties in vivo by examining RGC survival and axon regeneration at 4 weeks following intraorbital optic nerve transection in adult rat. EPO was administered as a single intravitreal injection at the time of transection (5, 10, 25, 50 units, PBS control). Intravitreal EPO (5, 10 units) significantly increased RGC somata and axon survival between the eye and transection site. Twenty five units did not improve survival of RGC somata but did increase axon survival between the eye and transection site. In addition, a small proportion of axons penetrated the transection site and regenerated up to 1 mm into the distal nerve. In a second series, intravitreal EPO (25 units) doubled the number of RGC axons regenerating along a length of peripheral nerve grafted onto the retrobulbar optic nerve. Our in vivo evidence of both neuroregeneration and neuroprotection, taken together with the natural occurrence of EPO within the body and its ability to cross the blood-brain barrier, suggests that it offers promise as a therapeutic agent for central nerve repair.

Graded ephrin-A2 expression in the developing hamster superior colliculus.

During development, ephrin gradients guide retinal ganglion cell axons to their appropriate topographic locations in the superior colliculus (SC). Expression of ephrin-A2, assessed immunohistochemically in the developing hamster SC, revealed a rostral(low) to caudal (high) gradient that is most prominent at postnatal days P4 and P7 when topography is established. Double-labelling immunohistochemistry for ephrin-A2 and cell specific markers revealed that ephrin-A2 is expressed exclusively by a subset of neurons. The expression pattern has implications for mechanisms underlying establishment of topography during development and following injury.

Transient up-regulation of retinal EphA3 and EphA5, but not ephrin-A2, coincides with re-establishment of a topographic map during optic nerve regeneration in goldfish.

Eph tyrosine kinase receptors and their ligands, the ephrins, play a key role in the establishment of retinotectal topography during development. Tectal up-regulation of ephrin-A2 in goldfish, coincident with the reestablishment of a retinotectal map, suggests a similar role during optic nerve regeneration. Here we report a complementary study of EphA3, EphA5 and ephrin-A2 expression in the retina. EphA3 and EphA5 are transiently up-regulated as ascending naso-temporal gradients, whereas ephrin-A2 remains uniform. The expression profiles differ from those in developing chick and mouse, suggesting that different combinations of retinal Eph receptors and ligands can generate topographic guidance information.