Erythropoietin either Prevents or Exacerbates Retinal Damage from Eye Trauma Depending on Treatment Timing.

PURPOSE: Erythropoietin (EPO) is a promising neuroprotective agent and is currently in Phase III clinical trials for the treatment of traumatic brain injury. The goal of this study was to determine if EPO is also protective in traumatic eye injury. METHODS: The left eyes of anesthetized DBA/2J or Balb/c mice were exposed to a single 26 psi overpressure air-wave while the rest of the body was shielded. DBA/2J mice were given intraperitoneal injections of EPO or buffer and analyses were performed at 3 or 7 days post-blast. Balb/c mice were given intramuscular injections of rAAV.EpoR76E or rAAV.eGFP either pre- or post-blast and analyses were performed at 1 month post-blast. RESULTS: EPO had a bimodal effect on cell death, glial reactivity, and oxidative stress. All measures were increased at 3 days post-blast and decreased at 7-days post-blast. Increased retinal ferritin and NADPH oxygenases were detected in retinas from EPO-treated mice. The gene therapy approach protected against axon degeneration, cell death, and oxidative stress when given after blast, but not before. CONCLUSIONS: Systemic, exogenous EPO and EPO-R76E protects the retina after trauma even when initiation of treatment is delayed by up to 3 weeks. Systemic treatment with EPO or EPO-R76E beginning before or soon after trauma may exacerbate protective effects of EPO within the retina as a result of increased iron levels from erythropoiesis and, thus, increased oxidative stress within the retina. This is likely overcome with time as a result of an increase in levels of antioxidant enzymes. Either intraocular delivery of EPO or treatment with non-erythropoietic forms of EPO may be more efficacious.

Exacerbation of blast-induced ocular trauma by an immune response.

BACKGROUND: Visual prognosis after an open globe injury is typically worse than after a closed globe injury due, in part, to the immune response that ensues following open globe trauma. There is a need for an animal model of open globe injury in order to investigate mechanisms of vision loss and test potential therapeutics. METHODS: The left eyes of DBA/2 J mice were exposed to an overpressure airwave blast. This strain lacks a fully functional ocular immune privilege, so even though the blast wave does not rupture the globe, immune infiltrate and neuroinflammation occurs as it would in an open globe injury. For the first month after blast wave exposure, the gross pathology, intraocular pressure, visual function, and retinal integrity of the blast-exposed eyes were monitored. Eyes were collected at three, seven, and 28 days to study the histology of the cornea, retina, and optic nerve, and perform immunohistochemical labeling with markers of cell death, oxidative stress, and inflammation. RESULTS: The overpressure airwave caused anterior injuries including corneal edema, neovascularization, and hyphema. Immune infiltrate was detected throughout the eyes after blast wave exposure. Posterior injuries included occasional retinal detachments and epiretinal membranes, large retinal pigment epithelium vacuoles, regional photoreceptor cell death, and glial reactivity. Optic nerve degeneration was evident at 28 days post-blast wave exposure. The electroretinogram (ERG) showed an early deficit in the a wave that recovered over time. Both visual acuity and the ERG b wave showed an early decrease, then a transient improvement that was followed by further decline at 28 days post-blast wave exposure. CONCLUSIONS: Ocular blast injury in the DBA/2 J mouse recapitulates damage that is characteristic of open globe injuries with the advantage of a physically intact globe that prevents complications from infection. The injury was more severe in DBA/2 J mice than in C57Bl/6 J mice, which have an intact ocular immune privilege. Early injury to the outer retina mostly recovers over time. In contrast, inner retinal dysfunction seems to drive later vision loss.

Motor, visual and emotional deficits in mice after closed-head mild traumatic brain injury are alleviated by the novel CB2 inverse agonist SMM-189.

We have developed a focal blast model of closed-head mild traumatic brain injury (TBI) in mice. As true for individuals that have experienced mild TBI, mice subjected to 50-60 psi blast show motor, visual and emotional deficits, diffuse axonal injury and microglial activation, but no overt neuron loss. Because microglial activation can worsen brain damage after a concussive event and because microglia can be modulated by their cannabinoid type 2 receptors (CB2), we evaluated the effectiveness of the novel CB2 receptor inverse agonist SMM-189 in altering microglial activation and mitigating deficits after mild TBI. In vitro analysis indicated that SMM-189 converted human microglia from the pro-inflammatory M1 phenotype to the pro-healing M2 phenotype. Studies in mice showed that daily administration of SMM-189 for two weeks beginning shortly after blast greatly reduced the motor, visual, and emotional deficits otherwise evident after 50-60 psi blasts, and prevented brain injury that may contribute to these deficits. Our results suggest that treatment with the CB2 inverse agonist SMM-189 after a mild TBI event can reduce its adverse consequences by beneficially modulating microglial activation. These findings recommend further evaluation of CB2 inverse agonists as a novel therapeutic approach for treating mild TBI.

A Novel Closed-Head Model of Mild Traumatic Brain Injury Using Focal Primary Overpressure Blast to the Cranium in Mice.

Mild traumatic brain injury (TBI) from focal head impact is the most common form of TBI in humans. Animal models, however, typically use direct impact to the exposed dura or skull, or blast to the entire head. We present a detailed characterization of a novel overpressure blast system to create focal closed-head mild TBI in mice. A high-pressure air pulse limited to a 7.5 mm diameter area on the left side of the head overlying the forebrain is delivered to anesthetized mice. The mouse eyes and ears are shielded, and its head and body are cushioned to minimize movement. This approach creates mild TBI by a pressure wave that acts on the brain, with minimal accompanying head acceleration-deceleration. A single 20-psi blast yields no functional deficits or brain injury, while a single 25-40 psi blast yields only slight motor deficits and brain damage. By contrast, a single 50-60 psi blast produces significant visual, motor, and neuropsychiatric impairments and axonal damage and microglial activation in major fiber tracts, but no contusive brain injury. This model thus reproduces the widespread axonal injury and functional impairments characteristic of closed-head mild TBI, without the complications of systemic or ocular blast effects or head acceleration that typically occur in other blast or impact models of closed-skull mild TBI. Accordingly, our model provides a simple way to examine the biomechanics, pathophysiology, and functional deficits that result from TBI and can serve as a reliable platform for testing therapies that reduce brain pathology and deficits.

Identification of a therapeutic dose of continuously delivered erythropoietin in the eye using an inducible promoter system.

Erythropoietin (EPO) can protect the retina from acute damage, but long-term systemic treatment induces polycythemia. Intraocular gene delivery of EPO is not protective despite producing high levels of EPO likely due to its bellshaped dose curve. The goal of this study was to identify a therapeutic dose of continuously produced EPO in the eye. We packaged a mutated form of EPO (EPOR76E) that has equivalent neuroprotective activity as wild-type EPO and attenuated erythropoietic activity into a recombinant adeno-associated viral vector under the control of the tetracycline inducible promoter. This vector was injected into the subretinal space of homozygous postnatal 5-7 day retinal degeneration slow mice, that express the tetracycline transactivators from a retinal pigment epithelium specific promoter. At weaning, mice received a single intraperitoneal injection of doxycycline and were then maintained on water with or without doxycycline until postnatal day 60. Intraocular EPO levels and outer nuclear layer thickness were quantified and correlated. Control eyes contained 6.1 ± 0.1 (SEM) mU/ml EPO. The eyes of mice that received an intraperitoneal injection of doxycycline contained 11.8 ± 2.0 (SEM) mU/ml EPO-R76E. Treatment with doxycycline water induced production of 35.9 ± 2.4 (SEM) mU/ml EPO-R76E in the eye. The outer nuclear layer was approximately 8 µm thicker in eyes of mice that received doxycycline water as compared to the control groups. Our data indicates that drug delivery systems should be optimized to deliver at least 36 mU/ml EPO into the eye since this dose was effective for the treatment of a progressive retinal degeneration.