Manda Saraswati

Physiologic progesterone reduces mitochondrial dysfunction and hippocampal cell loss after traumatic brain injury in female rats.

Growing literature suggests important sex-based differences in outcome following traumatic brain injury (TBI) in animals and humans. Progesterone has emerged as a key hormone involved in many potential neuroprotective pathways after acute brain injury and may be responsible for some of these differences. Many studies have utilized supraphysiologic levels of post-traumatic progesterone to reverse pathologic processes after TBI, but few studies have focused on the role of endogenous physiologic levels of progesterone in neuroprotection. We hypothesized that progesterone at physiologic serum levels would be neuroprotective in female rats after TBI and that progesterone would reverse early mitochondrial dysfunction seen in this model. Female, Sprague-Dawley rats were ovariectomized and implanted with silastic capsules containing either low or high physiologic range progesterone at 7 days prior to TBI. Control rats received ovariectomy with implants containing no hormone. Rats underwent controlled cortical impact to the left parietotemporal cortex and were evaluated for evidence of early mitochondrial dysfunction (1 h) and delayed hippocampal neuronal injury and cortical tissue loss (7 days) after injury. Progesterone in the low physiologic range reversed the early postinjury alterations seen in mitochondrial respiration and reduced hippocampal neuronal loss in both the CA1 and CA3 subfields. Progesterone in the high physiologic range had a more limited pattern of hippocampal neuronal preservation in the CA3 region only. Neither progesterone dose significantly reduced cortical tissue loss. These findings have implications in understanding the sex-based differences in outcome following acute brain injury.

Mitochondrial dysfunction early after traumatic brain injury in immature rats

Mitochondria play central roles in acute brain injury; however, little is known about mitochondrial function following traumatic brain injury (TBI) to the immature brain. We hypothesized that TBI would cause mitochondrial dysfunction early (<4 h) after injury. Immature rats underwent controlled cortical impact (CCI) or sham injury to the left cortex, and mitochondria were isolated from both hemispheres at 1 and 4 h after TBI. Rates of phosphorylating (State 3) and resting (State 4) respiration were measured with and without bovine serum albumin. The respiratory control ratio was calculated (State 3/State 4). Rates of mitochondrial H2O2 production, pyruvate dehydrogenase complex enzyme activity, and cytochrome c content were measured. Mitochondrial State 4 rates (ipsilateral/contralateral ratios) were higher after TBI at 1 h, which was reversed with bovine serum albumin. Four hours after TBI, pyruvate dehydrogenase complex activity and cytochrome c content (ipsilateral/contralateral ratios) were lower in TBI mitochondria. These data demonstrate abnormal mitochondrial function early (≤4 h) after TBI in the developing brain. Future studies directed at reversing mitochondrial abnormalities could guide neuroprotective interventions after pediatric TBI.

Cyclosporin A Preserves Mitochondrial Function after Traumatic Brain Injury in the Immature Rat and Piglet

Cyclosporin A (CsA) has been shown to be neuroprotective in mature animal models of traumatic brain injury (TBI), but its effects on immature animal models of TBI are unknown. In mature animal models, CsA inhibits the opening of the mitochondrial permeability transition pore (MPTP), thereby maintaining mitochondrial homeostasis following injury by inhibiting calcium influx and preserving mitochondrial membrane potential. The aim of the present study was to evaluate CsAs ability to preserve mitochondrial bioenergetic function following TBI (as measured by mitochondrial respiration and cerebral microdialysis), in two immature models (focal and diffuse), and in two different species (rat and piglet). Three groups were studied: injured+CsA, injured+saline vehicle, and uninjured shams. In addition, we evaluated CsAs effects on cerebral hemodynamics as measured by a novel thermal diffusion probe. The results demonstrate that post-injury administration of CsA ameliorates mitochondrial dysfunction, preserves cerebral blood flow (CBF), and limits neuropathology in immature animals 24 h post-TBI. Mitochondria were isolated 24 h after controlled cortical impact (CCI) in rats and rapid non-impact rotational injury (RNR) in piglets, and CsA ameliorated cerebral bioenergetic crisis with preservation of the respiratory control ratio (RCR) to sham levels. Results were more dramatic in RNR piglets than in CCI rats. In piglets, CsA also preserved lactate pyruvate ratios (LPR), as measured by cerebral microdialysis and CBF at sham levels 24 h after injury, in contrast to the significant alterations seen in injured piglets compared to shams (p<0.01). The administration of CsA to piglets following RNR promoted a 42% decrease in injured brain volume (p<0.01). We conclude that CsA exhibits significant neuroprotective activity in immature models of focal and diffuse TBI, and has exciting translational potential as a therapeutic agent for neuroprotection in children.

Early and Sustained Alterations in Cerebral Metabolism after Traumatic Brain Injury in Immature Rats

Although studies have shown alterations in cerebral metabolism after traumatic brain injury (TBI), clinical data in the developing brain is limited. We hypothesized that post-traumatic metabolic changes occur early (<24 h) and persist for up to 1 week. Immature rats underwent TBI to the left parietal cortex. Brains were removed at 4 h, 24 h, and 7 days after injury, and separated into ipsilateral (injured) and contralateral (control) hemispheres. Proton nuclear magnetic resonance (NMR) spectra were obtained, and spectra were analyzed for N-acetyl-aspartate (NAA), lactate (Lac), creatine (Cr), choline, and alanine, with metabolite ratios determined (NAA/Cr, Lac/Cr). There were no metabolic differences at any time in sham controls between cerebral hemispheres. At 4 and 24 h, there was an increase in Lac/Cr, reflecting increased glycolysis and/or decreased oxidative metabolism. At 24 h and 7 days, there was a decrease in NAA/Cr, indicating loss of neuronal integrity. The NAA/Lac ratio was decreased ( approximately 15-20%) at all times (4 h, 24 h, 7 days) in the injured hemisphere of TBI rats. In conclusion, metabolic derangements begin early (<24 h) after TBI in the immature rat and are sustained for up to 7 days. Evaluation of early metabolic alterations after TBI could identify novel targets for neuroprotection in the developing brain.

Immunolocalization of Bex protein in the mouse brain and olfactory system.

Bex proteins are expressed from a family of “brain expressed X‐linked genes” that are closely linked on the X‐chromosome. Bex1 and 2 have been characterized as interacting partners of the olfactory marker protein (OMP). Here we report the distribution of Bex1 and Bex2 mRNAs in several brain regions and the development and characterization of an antibody to mouse Bex1 protein that cross‐reacts with Bex2 (but not Bex3), and its use to determine the cellular distribution of Bex proteins in the murine brain. The specificity of the antiserum was characterized by immunoprecipitation and Western blots of tissue and transfected cell extracts and by immunocytochemical analyses of cells transfected with either Bex1 or Bex2. Antibodies preabsorbed with Bex2 still recognize Bex1, while blocking with Bex1 eliminates all immunoreactivity to both Bex1 and Bex2. Bex immunoreactivity (ir) was primarily localized to neuronal cells within several regions of the brain, including the olfactory epithelium, bulb, peri/paraventricular nuclei, suprachiasmatic nucleus, arcuate nucleus, median eminence, lateral hypothalamic area, thalamus, hippocampus, and cerebellum. RT‐PCR and in situ hybridization demonstrated the presence of Bex mRNA in several of these regions. Double‐label immunocytochemistry indicates that Bex‐ir is colocalized with OMP in mature olfactory receptor neurons (ORNs) and in the OMP‐positive subpopulation of neurons in hypothalamus. This is the first anatomical mapping of Bex proteins in the mouse brain and their colocalization with OMP in ORNs and hypothalamus. J. Comp. Neurol. 487:1–14, 2005.

A New Rabbit Model of Pediatric Traumatic Brain Injury

Traumatic brain injury (TBI) is a common cause of disability in childhood, resulting in numerous physical, behavioral, and cognitive sequelae, which can influence development through the lifespan. The mechanisms by which TBI influences normal development and maturation remain largely unknown. Pediatric rodent models of TBI often do not demonstrate the spectrum of motor and cognitive deficits seen in patients. To address this problem, we developed a New Zealand white rabbit model of pediatric TBI that better mimics the neurological injury seen after TBI in children. On postnatal Day 5-7 (P5-7), rabbits were injured by a controlled cortical impact (6-mm impactor tip; 5.5 m/sec, 2-mm depth, 50-msec duration). Rabbits from the same litter served as naïve (no injury) and sham (craniotomy alone) controls. Functional abilities and activity levels were measured 1 and 5 d after injury. Maturation level was monitored daily. We performed cognitive tests during P14-24 and sacrificed the animals at 1, 3, 7, and 21 d after injury to evaluate lesion volume and microglia. TBI kits exhibited delayed achievement of normal developmental milestones. They also demonstrated significant cognitive deficits, with lower percentage of correct alternation rate in the T-maze (n=9-15/group; p<0.001) and less discrimination between novel and old objects (p<0.001). Lesion volume increased from 16% at Day 3 to 30% at Day 7 after injury, indicating ongoing secondary injury. Activated microglia were noted at the injury site and also in white matter regions of the ipsilateral and contralateral hemispheres. The neurologic and histologic changes in this model are comparable to those reported clinically. Thus, this rabbit model provides a novel platform for evaluating neuroprotective therapies in pediatric TBI.

769: SEX DIFFERENCES IN RESPONSE TO 20-HETE INHIBITION AFTER TRAUMATIC BRAIN INJURY IN IMMATURE RATS

Learning Objectives: Emerging evidence suggests that metabolites of the arachidonic acid pathway play important roles in neuronal and astrocyte signaling. Previous work has shown that inhibition of 20-hydroxyeicosatetraenoic acid (20-HETE) formation by cytochrome P450 (CYP) omega-hydoxylation of arachidonic acid can protect immature and mature brain from ischemia. However, the majority of studies have been performed in adult, male animals. We tested the hypothesis that post-treatment with the 20-HETE synthesis inhibitor N-hydroxy-N-4-butyl-2-methylphenylformamidine (HET0016) can be neuroprotective after pediatric TBI in both male and female rat pups. Methods: Male and female Sprague Dawley rats (postnatal day 9–10) were subjected to controlled cortical impact (CCI; 3 mm impactor; velocity 5.5 m/s; depth 1.5 mm), and studied in 3 groups: 1) vehicle-treated sham, 2) vehicle-treated TBI, and 3) HET0016-treated TBI (1 mg/kg, ip, at 5 min and 3 h post-injury). At 30d after CCI, rats underwent neurologic testing (foot fault, novel object recognition/NOR), and percent tissue loss in the ipsilateral hemisphere was measured. Data are presented as mean ± SEM, and were analyzed by AVOVA. Results: In male rats, the number of contralateral hindlimb foot faults were reduced (TBI+veh=8.9 ± 1.1; TBI+HET=4.2 ± 0.9) and the discrimination index on NOR was improved (TBI+veh=0.37 ± 0.08; TBI+HET=0.50 ± 0.07) after treatment with HET0016. Also, HET0016 reduced the percent tissue loss after TBI in male rats. In contrast, there were no differences between groups in performance on foot fault or NOR testing in female rats, and HET0016 did not change the percent tissue loss in female rats. Conclusions: The 20-HETE synthesis inhibitor HET0016 improved both histologic and neurologic outcome after pediatric TBI in male rats, but not in female rats. This suggests potentially important sex differences in the response to treatment after pediatric TBI. Future studies will be directed at understanding sex differences in the pathobiologic response to injury in the developing brain, specifically targeting the role of 20-HETE in cell death after TBI.

548: THE EFFECT OF PROGESTERONE ON MICROGLIAL ACTIVATION IN A RAT MODEL OF PEDIATRIC TBI

Learning Objectives: Traumatic brain injury (TBI), the leading cause of disability and death in children in the U.S., results in over 2600 deaths annually. There are limited data as to how to improve outcome after pediatric TBI. Progesterone is a neurosteroid that is synthesized in the CNS and previous work in our laboratory has shown that progesterone prevents mitochondrial dysfunction and reduces lesion volume after TBI in immature rats. There is little existing data to quantify the anti-inflammatory effects of progesterone after injury. Microglia are responsible for homeostasis and repair in healthy and injured brain tissue. In this study we hypothesize that progesterone will decrease the number of activated microglia in a rat model of pediatric TBI. Methods: Sprague-Dawley rats (PND 10 & 17) underwent controlled cortical impact (CCI) to the left parietal cortex, with control rats undergoing sham craniotomy. Rats were assigned to receive progesterone (10 mg/kg IP at 1h, SQ at 6h and repeated every 24h) or vehicle (22.5% cyclodextrin administered at the same time). Rats were sacrificed 7 days after injury and sections of ipsilateral and contralateral thalamus were examined. Specimens were stained with IBA1 and evaluated under 20X magnification. Microglia were manually counted and qualified by a blinded observer. Results:PND 10 males had 84.2% activation after TBI. After progesterone, activated microglia were decreased by 20%, although the total number of microglia was increased. In PND 17 males, 90.1% of microglia were activated after TBI. There was a 15% decrease in activated microglia and almost 40% decrease in total amount of microglia after progesterone therapy in this group. There was no difference between female groups after progesterone therapy. Conclusions: Progesterone reduced the percent of activated microglia present in male rats after TBI. Additionally, progesterone reduced the overall number of microglia in PND 17 males. There was no difference in females after progesterone therapy. These sex and age differences will be significant as we move toward clinical trials of progesterone after pediatric TBI.