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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% (p0.05), decreased apoptosis by 13% (p0.05) and promoted keratinocyte migration by up to 14% (p0.05), 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 (p0.05). 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, p0.05) and reduced epidermal thickness (MT-I/II: 45+/-4 microm vs. PBS: 101+/-19 microm, p0.05) 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.

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.

Rat retinal ganglion cells (RGCs) are generated between embryonic day (E) 13 and E19. Retinal axons first reach the superior colliculus at E16/16.5 but the time of arrival of axons from late-born RGCs is unknown. This study examined (i) whether there is a correlation between RGC genesis and the timing of retinotectal innervation and (ii) when axons of late-born RGCs reach the superior colliculus. Pregnant Wistar rats were injected intraperitoneally with bromodeoxyuridine (BrdU) on E16, E18 or E19. Pups from these litters received unilateral superior colliculus injections of fluorogold (FG) at ages between postnatal (P) day P0 and P6, and were perfused 1-2 days later. RGCs in 3 rats from each BrdU litter were labelled in adulthood by placing FG onto transected optic nerve. Retinas were cryosectioned and the number of FG, BrdU and double-labelled (FG+/BrdU+) RGCs quantified. In the E16 group, the proportion of FG-labelled RGCs that were BrdU+ did not vary with age, indicating that axons from these cells had reached the superior colliculus by P0/P1. In contrast, for the smaller cohorts of RGCs born on E18 or E19, the proportion of BrdU+ cells that were FG+ increased significantly after birth; axons from most RGCs born on E19 were not retrogradely FG-labelled until P4/P5. Thus there is a correlation between birthdate and innervation in rat retinotectal pathways. Furthermore, compared to the earliest born RGCs, axons from late-born RGCs take about three times longer to reach the superior colliculus. Later-arriving axons presumably encounter comparatively different growth terrains en route and eventually innervate more differentiated target structures.

A single course of corticosteroid reduces intracranial hemorrhage in preterm infants. The mechanism of protection is unclear. Glial fibrillary acidic protein (GFAP), expressed by astrocytes, is regulated by glucosteroids and is an important component of the cells forming the blood brain barrier. We have evaluated the effect of prenatal corticosteroid upon ovine GFAP.Date-mated ewes were studied in two protocols and lambs delivered on day 125 or 145 (term = 150). In the maternal injection protocol (n = 36) ewes were administered saline, single or repeated injections of corticosteroid. In the fetal injection protocol (n = 48) direct ultrasound-guided fetal injections of saline, single or repeated corticosteroid were administered, and an additional control group did not receive fetal injections. Optic nerve GFAP immunohistochemistry was performed and quantified.At 125 days, repeated, but not single, administration of corticosteroid, by either maternal or fetal route, was associated with a significant reduction in GFAP (both p0.002); by 145 days, the deficit had recovered (both p0.05). The process of performing repeated fetal injections had an independent effect upon GFAP at 145 days (p = 0.002).Repeated administration of corticosteroid results in a reduction in GFAP in the developing ovine optic nerve, with recovery demonstrated by 145 days.

In this chapter we describe aspects of the changing morphology of retinal ganglion cells during both normal development and regeneration. We labeled cells with the neuronal tracer horseradish peroxidase (HRP) to visualize the dendritic trees or the axons of individual cells. This approach has allowed us to gain insights into the morphology of cell processes beyond that afforded by an examination of the cell population as a whole. We have examined the development of dendrites in the quokka (a type of wallaby or small kangaroo) and of axons in the developing optic nerve of the chick, quokka, rabbit, and cat. We have also examined the morphology of optic axons in frog as they regenerate through the site of an optic nerve crush.