Noninvasive magnetic resonance imaging stratifies injury severity in a rodent model of male juvenile traumatic brain injury.

Age and severity are significant predictors of traumatic brain injury (TBI) outcomes in the immature brain. TBI studies have segregated TBI injury into three severity groups: mild, moderate, and severe. While mild TBI is most frequent form in children and adults, there is debate over the indicators used to denote mild injury. Clinically, magnetic resonance imaging (MRI) and computed tomography (CT) are used to diagnose the TBI severity when medically warranted. Herein, we induced mild, moderate, and severe TBI in juvenile rats (jTBI) using the controlled cortical impact model. We characterized the temporal and spatial injury after graded jTBI in vivo using high-field MRI at 0.25 (6 hr), 1 and 3 days post-injury (dpi) with comparative histology. Susceptibility-weighted imaging (SWI) for blood and T2-weighted imaging (T2WI) for edema were quantified over the 0.25-3 dpi. Edema volumes increased linearly with severity at 0.25 dpi that slowly continued to decrease over the 3 dpi. In contrast, blood volumes did not decrease over time. Mild TBI had the least amount of blood visible on SWI. Fluoro-jade B (FJB) staining for cell death confirmed increased cellular death with increasing severity and increased FJB + cells in the corpus callosum (CC). Interestingly, the strongest correlation was observed for cell death and the presence of extravascular blood. A clear understanding of acute brain injury (jTBI) and how blood/edema contribute to mild, moderate, and severe jTBI is needed prior to embarking on therapeutic interventions. Noninvasive imaging should be used in mild jTBI to verify lack of overt injury.

Development of MGD007, a gpA33 x CD3-Bispecific DART Protein for T-Cell Immunotherapy of Metastatic Colorectal Cancer.

We have developed MGD007 (anti-glycoprotein A33 x anti-CD3), a DART protein designed to redirect T cells to target gpA33 expressing colon cancer. The gpA33 target was selected on the basis of an antibody-based screen to identify cancer antigens universally expressed in both primary and metastatic colorectal cancer specimens, including putative cancer stem cell populations. MGD007 displays the anticipated-bispecific binding properties and mediates potent lysis of gpA33-positive cancer cell lines, including models of colorectal cancer stem cells, through recruitment of T cells. Xenograft studies showed tumor growth inhibition at doses as low as 4 µg/kg. Both CD8 and CD4 T cells mediated lysis of gpA33-expressing tumor cells, with activity accompanied by increases in granzyme and perforin. Notably, suppressive T-cell populations could also be leveraged to mediate lysis of gpA33-expressing tumor cells. Concomitant with CTL activity, both T-cell activation and expansion are observed in a gpA33-dependent manner. No cytokine activation was observed with human PBMC alone, consistent with the absence of gpA33 expression on peripheral blood cell populations. Following prolonged exposure to MGD007 and gpA33 positive tumor cells, T cells express PD-1 and LAG-3 and acquire a memory phenotype but retain ability to support potent cell killing. In cynomolgus monkeys, 4 weekly doses of 100 µg/kg were well tolerated, with prolonged PK consistent with that of an Fc-containing molecule. Taken together, MGD007 displays potent activity against colorectal cancer cells consistent with a mechanism of action endowed in its design and support further investigation of MGD007 as a potential novel therapeutic treatment for colorectal cancer. Mol Cancer Ther; 17(8); 1761-72. ©2018 AACR.

Posttraumatic reduction of edema with aquaporin-4 RNA interference improves acute and chronic functional recovery.

Traumatic brain injury (TBI) is common in young children and adolescents and is associated with long-term disability and mortality. The neuropathologic sequelae that result from juvenile TBI are a complex cascade of events that include edema formation and brain swelling. Brain aquaporin-4 (AQP4) has a key role in edema formation. Thus, development of novel treatments targeting AQP4 to reduce edema could lessen the neuropathologic sequelae. We hypothesized that inhibiting AQP4 expression by injection of small-interfering RNA (siRNA) targeting AQP4 (siAQP4) after juvenile TBI would decrease edema formation, neuroinflammation, neuronal cell death, and improve neurologic outcomes. The siAQP4 or a RNA-induced silencing complex (RISC)-free control siRNA (siGLO) was injected lateral to the trauma site after controlled cortical impact in postnatal day 17 rats. Magnetic resonance imaging, neurologic testing, and immunohistochemistry were performed to assess outcomes. Pups treated with siAQP4 showed acute (3 days after injury) improvements in motor function and in spatial memory at long term (60 days after injury) compared with siGLO-treated animals. These improvements were associated with decreased edema formation, increased microglial activation, decreased blood-brain barrier disruption, reduced astrogliosis and neuronal cell death. The effectiveness of our treatment paradigm was associated with a 30% decrease in AQP4 expression at the injection site.

Traumatic brain injury in young rats leads to progressive behavioral deficits coincident with altered tissue properties in adulthood.

Traumatic brain injury (TBI) affects many infants and children, and results in enduring motor and cognitive impairments with accompanying changes in white matter tracts, yet few experimental studies in rodent juvenile models of TBI (jTBI) have examined the timeline and nature of these deficits, histologically and functionally. We used a single controlled cortical impact (CCI) injury to the parietal cortex of rats at post-natal day (P) 17 to evaluate behavioral alterations, injury volume, and morphological and molecular changes in gray and white matter, with accompanying measures of electrophysiological function. At 60 days post-injury (dpi), we found that jTBI animals displayed behavioral deficits in foot-fault and rotarod tests, along with a left turn bias throughout their early developmental stages and into adulthood. In addition, anxiety-like behaviors on the zero maze emerged in jTBI animals at 60 dpi. The final lesion constituted only ∼3% of brain volume, and morphological tissue changes were evaluated using MRI, as well as immunohistochemistry for neuronal nuclei (NeuN), myelin basic protein (MBP), neurofilament-200 (NF200), and oligodendrocytes (CNPase). White matter morphological changes were associated with a global increase in MBP immunostaining and reduced compound action potential amplitudes at 60 dpi. These results suggest that brain injury early in life can induce long-term white matter dysfunction, occurring in parallel with the delayed development and persistence of behavioral deficits, thus modeling clinical and longitudinal TBI observations.

Brain irradiation improves focal cerebral ischemia recovery in aged rats.

BACKGROUND: Studies have shown that aging is a significant factor in worsening stroke outcomes. While many mechanisms may aggravate brain injury in the elderly, one such potential system may involve increased glial proliferation in the aged stroke patient that could result in increased scar formation. We hypothesized that in aged rats a single brain-only exposure to a low radiation dose prior to focal brain ischemia would reduce glial proliferation and confer a long-term neuroprotective effect. METHODS: Brain-only proton irradiation (8 Gy) was performed ten days prior to middle cerebral artery occlusion (MCAO) in aged male rats. Magnetic resonance imaging (MRI) was undertaken in naive, radiation-only (Rad), MCAO, and MCAO+Rad groups at 2, 14 and 28 days post-stroke followed by immunohistochemistry (day 28). RESULTS: Ischemic lesion volume in MCAO+Rad group was decreased by 50.7% with an accelerated temporal reduction in peri-lesional brain edema and increased water mobility within the ischemic core (39.8%) compared to MCAO-only rats. In the peri-lesional brain region of MCAO+Rad rats there was a decreased scar formation (49%, glial fibrillary acidic protein), brain tissue sclerosis (30%, aquaporin-4) and necrosis/apoptosis (58%, TUNEL positive cells) compared to those in MCAO animals. CONCLUSION: In aged animals a single exposure to brain-only radiation prior to focal cerebral ischemia is neuroprotective as it prevents glial hyperproliferation, progressive brain tissue sclerosis and reduces the apoptosis/necrosis in the peri-lesional region. Decreased lesion volume is in agreement with accelerated reduction of brain edema in these animals.

Brain water mobility decreases after astrocytic aquaporin-4 inhibition using RNA interference.

Neuroimaging with diffusion-weighted imaging is routinely used for clinical diagnosis/prognosis. Its quantitative parameter, the apparent diffusion coefficient (ADC), is thought to reflect water mobility in brain tissues. After injury, reduced ADC values are thought to be secondary to decreases in the extracellular space caused by cell swelling. However, the physiological mechanisms associated with such changes remain uncertain. Aquaporins (AQPs) facilitate water diffusion through the plasma membrane and provide a unique opportunity to examine the molecular mechanisms underlying water mobility. Because of this critical role and the recognition that brain AQP4 is distributed within astrocytic cell membranes, we hypothesized that AQP4 contributes to the regulation of water diffusion and variations in its expression would alter ADC values in normal brain. Using RNA interference in the rodent brain, we acutely knocked down AQP4 expression and observed that a 27% AQP4-specific silencing induced a 50% decrease in ADC values, without modification of tissue histology. Our results demonstrate that ADC values in normal brain are modulated by astrocytic AQP4. These findings have major clinical relevance as they suggest that imaging changes seen in acute neurologic disorders such as stroke and trauma are in part due to changes in tissue AQP4 levels.

Rodent neonatal bilateral carotid artery occlusion with hypoxia mimics human hypoxic-ischemic injury.

We report a new clinically relevant model of neonatal hypoxic-ischemic injury in a 10-day-old rat pup. Bilateral carotid artery occlusion and 8% hypoxia (1 to 15 mins, BCAO-H) was induced with varying degrees of injury (mild, moderate, severe), which was quantified using magnetic resonance imaging including diffusion-weighted and T2-weighted imaging at 24 h and 21/28 days. We developed a magnetic resonance imaging-based rat pup severity score and compared 3D ischemic injury volumes/rat pup severity score with histology and behavioral testing. At 24 h, hypoxic-ischemic injury was observed in 17/27 animals; long-term survival was 81%. Magnetic resonance imaging lesion volumes did not correlate with hypoxia duration but correlated with rat pup severity score, which was used to classify animals into mild (n=21), moderate (n=6), and severe (n=10) groups with average brain lesion volumes of 0.9%, 33.2%, and 56.3%, respectively. Histology confirmed lesion location and histologic scoring correlated with the rat pup severity score. We also found excellent correlation between injury severity and multiple behavioral tasks. Bilateral carotid artery occlusion and hypoxia in the P10 rat pup is an excellent model of neonatal hypoxic-ischemic injury because it induces diffuse global injury similar to the term infant. This model can produce graded injury severity, similar to that seen in human neonates, but manipulation with hypoxia duration is unpredictable.