Ischemic Brain Injury
Ischemic brain injury caused by cardiac arrest and stroke kills approximately 300,000 people and disables another 150,000 each year in the United States. Despite decades of research, clinically effective therapies are limited to early reperfusion for stroke and therapeutic hypothermia after cardiac arrest. Unfortunately, these interventions benefit a small minority of patients. After an ischemic insult, neuronal death occurs over hours to days. This interval represents a therapeutic window within which neuroprotective strategies have the potential to significantly reduce brain damage and improve functional survival.
Traumatic Brain Injury
1.5 million Americans sustain a traumatic brain injury (TBI) every year. Of these, 50,000 will die and 80,000 of will suffer long-term disability due to permanent brain damage. Although important advances in patient care over the past few decades have improved survival after severe TBI, specific neuroprotective strategies have yet to be translated from pre-clinical models to clinical practice. As with ischemic brain injury, there is tremendous potential for neuroprotective strategies to reduce brain damage after head trauma.
The goal of the Brain Resuscitation Laboratory is to characterize the molecular events that cause neuronal death after acute brain injury and develop clinically effective therapies to minimize brain damage after cardiac arrest, stroke and head trauma. Current areas of focus are listed below
Neuronal Apoptosis and Necrosis after Global Brain Ischemia
One of our fundamental observations following global brain ischemia is that different neurons in different brain regions die by different mechanisms over different time courses. Figure 1 illustrates the rat hippocampus 72 hours after transient forebrain ischemia in which apoptotic granule neurons in the dentate gyrus are seen along with necrotic pyramidal neurons in the CA1 sector. This observation underscores the complex nature of acute brain injury in vivo.
Proteolytic Injury Cascades in Post-Ischemic Neurons
One of our major lines of investigation involves analyzing pathological protease activity in post-ischemic neurons. Both calpain and caspase family proteases are activated in the brain after ischemia and reperfusion, and their relative activity appears to determine whether neurons die by apoptosis or necrosis. For example caspase activity is detected within 30 minute of global brain ischemia in granule neurons of the rostral dentate gyrus that subsequently die by apoptosis (Figure 2, A & B). This rapid progression to the execution phase of apoptosis suggests a pathway independent of new gene expression. In contrast, calpain activity is detected between 24 and 48 hours after global brain ischemia in CA1 sector pyramidal neurons that eventually die by delayed necrosis (Figure 2, C & D). These examples further highlight the complexity of in vivo acute brain injury mechanism as well as the challenges to developing clinically effective interventions.
In Vivo Molecular Biology
The complex injury cascades that occur in adult neurons after acute brain injury cannot be accurately reproduced in vitro or in cell culture. On the other hand, mechanistic interpretation of in vivo studies is generally limited by the poor specificity of pharmacologic interventions. To overcome these obstacles, we are developing an in vivo molecular biology approach that utilizes viral vectors to manipulate gene expression in adult neurons. This approach allows us to overexpress or knock down (using shRNA) individual molecular components of acute injury cascades and then evaluate the impact on neurodegeneration following acute brain injury. Figure 3 illustrates an example where AAV2/5 vector engineered to express enhanced green fluorescent protein (EGFP) was used to transduce CA1 sector pyramidal neurons of rat hippocampus in vivo.
Axonal Degeneration after Traumatic Brain Injury
Head trauma causes mechanical stretch injury to the axons of the central nervous system resulting in disrupted axonal transport, focal swelling and eventually secondary axotomy. The clinical term for this phenomenon is diffuse axonal injury (DAI). An important characteristic of axonal stretch injury is rapid loss of microtubules (within 15 minutes). In regions where microtubules do not recover, axonal swellings develop, which are sites of organelle accumulation due to impaired axonal transport. Within several hours, secondary axotomy begins to occur at the site of axonal swellings. This delay in irreversible pathology suggests a potential therapeutic window for neuroprotective interventions.
Calpain activity has been detected within 15 minutes of DAI in animal models. The downstream consequences of early calpain activity after axonal stretch injury are poorly understood. Proteolysis of microtubule components as a mechanism for the persistent loss of microtubules is an intriguing possibility. Tubulin, tau, MAP1A, and MAP1B are known calpain substrates, and in vitro assays have demonstrated that calpain activity can cause both microtubule depolymerization and impaired repolymerization. These observations support our general hypothesis that early calpain activity after axonal stretch contributes to microtubule instability, impaired axonal transport, and subsequent secondary axotomy. We will test this hypothesis in vivo using a rat optic nerve stretch model that simulates the mechanics of DAI in humans. Viral vector mediated RNA interference will be used to manipulate the calpain system and allow us to determine the role of different calpain isoforms on axonal degeneration after stretch.