Early inflammatory mediator gene expression in two models of traumatic brain injury: ex vivo cortical slice in mice and in vivo cortical impact in piglets

Background: The immunological response during the first 24 hours after traumatic brain injury (TBI) may be a critical therapeutic interval for limiting the secondary neuronal damage that is influenced by enhanced inflammatory mediator expression. Methods: To gain further insight of the early injury response, we examined the expression of several inflammatory genes by real-time qPCR as a function of time or distance from injury in two distinct mammalian models: an ex vivo mouse cortical slice injury system and an in vivo piglet model of brain injury. Results: Interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), chemokine ligands 2 (CCL2), 3 (CCL3), 4 (CCL4), and prostaglandin-endoperoxide synthase 2 (PTGS2) mRNAs increased within 5 h after injury in mouse cortical slices. Chemokine and PTGS2 mRNAs remained elevated in slices at 24 h, whereas IL-1β and TNF-α expressions decreased from earlier peak levels. At 24 h after cortical injury in 1-month-old piglets, the expression of CCL2 mRNA was significantly increased in the lesion core and in the penumbra region. The expression of PTGS2, IL-1β, and TNF-α was variable among the piglets. Conclusions: These in vitro and large animal models of cortical injury expand our understanding of the early timing and spread of the immunological response and can serve as preclinical systems to facilitate the discovery of therapeutic agents for TBI aimed at regulating inflammatory mediator expression

Establishing a Clinically Relevant Large Animal Model Platform for TBI Therapy Development: Using Cyclosporin A as a Case Study

We have developed the first immature large animal translational treatment trial of a pharmacologic intervention for traumatic brain injury (TBI) in children. The preclinical trial design includes multiple doses of the intervention in two different injury types (focal and diffuse) to bracket the range seen in clinical injury and uses two post-TBI delays to drug administration. Cyclosporin A (CsA) was used as a case study in our first implementation of the platform because of its success in multiple preclinical adult rodent TBI models and its current use in children for other indications. Tier 1 of the therapy development platform assessed the short-term treatment efficacy after 24 h of agent administration. Positive responses to treatment were compared with injured controls using an objective effect threshold established prior to the study. Effective CsA doses were identified to study in Tier 2. In the Tier 2 paradigm, agent is administered in a porcine intensive care unit utilizing neurological monitoring and clinically relevant management strategies, and intervention efficacy is defined as improvement in longer term behavioral endpoints above untreated injured animals. In summary, this innovative large animal preclinical study design can be applied to future evaluations of other agents that promote recovery or repair after TBI

Mechanisms of reduced luteal sensitivity to PGF2alpha in ruminants.

The corpus luteum (CL) of ruminants exhibits periods of reduced sensitivity to the luteolysin prostaglandin F2α (PGF2α). The CL has reduced sensitivity to PGF2α early in the luteal phase. Mechanisms of reduced luteal sensitivity to PGF2α during the early portion of the luteal phase have been widely studied, with a focus on regulation of cyclooxygenase-2 and components of the endothelin system. The first ovulation in postpartum cows results in a CL that regresses during transition from a period of reduced sensitivity to susceptibility to PGF2α, and there is evidence that the first CL in the postpartum cow is more sensitive to PGF2α. Corpora lutea are less sensitive to PGF2α during the maternal recognition of pregnancy. In the ewe, studies were performed to quantify mRNA for prostaglandin metabolic enzymes and components of the endothelin system, as well as catabolism of PGF2α to inactive PGFM in CL expected to regress early in postpartum cows and in pregnant ewes expected to have CL that do not regress after an injection of PGF2α. In both experiments, transcription of enzymes involved in prostaglandin anabolism were regulated by the abundance of PGE synthase and PGF synthase mRNA instead of cyclooxygenase-2 mRNA. Catabolism of PGF2α to PGFM was regulated post-transcriptionally. It was also demonstrated that the endothelin system may contribute to reduced luteal sensitivity to PGF2α through alterations in the endothelin converting enzyme-1 mRNA instead of alterations in the abundance of preproendothelin-1 or endothelin receptor type A mRNA. In conclusion, the early regressing CL in the postpartum cow did not appear to differ from non-regressing CL with respect to PGE synthesis or PGF2α catabolism. Nevertheless, premature uterine secretion of PGF2α activates mediators of regression. In pregnant ewes, the conceptus, which alters uterine secretion of prostaglandins to a greater ratio of PGE2: PGF2α, may cause a similar shift in prostaglandin production in the CL to a greater ratio of PGE2: PGF2α, a reduction of endothelin converting enzyme-1, and greater catabolism of PGF2α to the inactive metabolite PGFM

Development of a Model of Hemispheric Hypodensity (“Big Black Brain”)

Subdural hematoma (SDH) is the most common finding after abusive head trauma (AHT). Hemispheric hypodensity (HH) is a radiological indicator of severe brain damage that encompasses multiple vascular territories, and may develop in the hemisphere(s) underlying the SDH. In some instances where the SDH is predominantly unilateral, the widespread damage is unilateral underlying the SDH. To date, no animal model has successfully replicated this pattern of injury. We combined escalating severities of the injuries and insults commonly associated with HH including SDH, impact, mass effect, seizures, apnea, and hypoventilation to create an experimental model of HH in piglets aged 1 week (comparable to human infants) to 1 month (comparable to human toddlers). Unilateral HH evolved over 24 h when kainic acid was applied ipsilateral to the SDH to induce seizures. Pathological examination revealed a hypoxic-ischemic injury-type pattern with vasogenic edema through much of the cortical ribbon with relative sparing of deep gray matter. The percentage of the hemisphere that was damaged was greater on the ipsilateral versus contralateral side and was positively correlated with SDH area and estimated seizure duration. Further studies are needed to parse out the pathophysiology of this injury and to determine if multiple injuries and insults act synergistically to induce a metabolic mismatch or if the mechanism of trauma induces severe seizures that drive this distinctive pattern of injury

Scaled Cortical Impact in Immature Swine: Effect of Age and Gender on Lesion Volume

The piglet scaled cortical impact model creates a focal contusion using a skull-mounted, spring-loaded blunt indentation device scaled to achieve identical tissue strains in subjects with different brain sizes. Preliminary data showed that contusion size increased proportional to subject age. This study details the results from a new, larger series of subjects of three ages, and compares the effect of age and additional host and physiologic variables on injury response. Sixty-seven subjects, including infant (5- to 7-day-old), “toddler” (1-month-old), and early adolescent (4-month-old) swine underwent scaled cortical impact under strict anesthetic protocols. Serum glucose, testosterone, and 17β-estradiol levels were measured. Lesion size was measured at 1 week post injury, as the ratio of the lesion area over the area of the contralateral hemisphere. Adolescent subjects had lesions over eight times larger than infants (p < 0.0001). Lesion volumes were larger in toddlers than in infants, most significantly for males (p < 0.05). Adolescent subjects were warmer on average, but there was no correlation between temperature and lesion volume within any age group. Serum glucose did not differ among ages. Infant males had the highest levels of circulating sex steroids. In this model, age was the most robust predictor of lesion size. Temperature had an effect, but did not explain all the variability seen among age groups. There was an interaction among gender, hormone levels, and lesion size in younger subjects. Characterization of these variables allows use of this model for treatment trials for subjects at different stages of maturation